Hydroxypropyl beta-cyclodextrin compositions and methods

ABSTRACT

This disclosure provides mixtures of beta-cyclodextrin molecules substituted at one or more hydroxyl positions by hydroxypropyl groups, the mixture optionally including unsubstituted beta-cyclodextrin molecules, for use as a pharmaceutically active ingredient; methods of making such mixtures; methods of qualifying such mixtures for use in a pharmaceutical composition suitable for intrathecal or intracerebroventricular administration; pharmaceutical compositions suitable for intrathecal or intracerebroventricular administration comprising such mixtures; and methods of using the pharmaceutical compositions for treatment of Niemann-Pick disease Type C.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/430,664, filed Jun. 4, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/372,899, filed Apr. 2, 2019, now U.S. Pat. No.10,709,730, which is a continuation of U.S. patent application Ser. No.16/134,028, filed Sep. 18, 2018, now U.S. Pat. No. 10,300,086, which isa continuation of U.S. patent application Ser. No. 15/499,831, filedApr. 27, 2017, now U.S. Pat. No. 10,258,641, which is a continuation ofU.S. patent application Ser. No. 15/288,876, filed Oct. 7, 2016, nowU.S. Pat. No. 9,675,634, which is a continuation of U.S. patentapplication Ser. No. 15/178,153, filed Jun. 9, 2016, now abandoned,which claims the benefit of U.S. Provisional Application Nos.62/345,721, filed Jun. 3, 2016; 62/331,385, filed May 3, 2016;62/314,765, filed Mar. 29, 2016; 62/308,736, filed Mar. 15, 2016;62/276,728, filed Jan. 8, 2016; 62/263,599, filed Dec. 4, 2015;62/249,876, filed Nov. 2, 2015; 62/245,974, filed Oct. 23, 2015;62/189,114, filed Jul. 6, 2015; 62/175,075, filed Jun. 12, 2015; and62/173,889, filed Jun. 10, 2015, each of which is incorporated in itsentirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was created in the performance of a Cooperative Researchand Development Agreement (Agreement Ref. No. 02947) with the NationalInstitutes of Health, an Agency of the Department of Health and HumanServices. The Government of the United States has certain rights in thisinvention.

BACKGROUND

Niemann-Pick disease Type C (NPC) is a lysosomal lipid storage disordercaused by autosomal recessive mutations in either the NPC1 or NPC2 gene.Symptoms typically manifest beginning in the perinatal period andprogress throughout life. The disorder often includes neurologicalsymptoms, such as cerebellar ataxia, dysarthria, seizures, vertical gazepalsy, motor impairment, dysphagia, psychotic episodes, and progressivedementia, as well as systemic symptoms in other organs, such as theliver, spleen, or lung. NPC has been described as a cellular cholesteroltransport defect, although in the brain accumulation of other lipids,such as GM2 and GM3 gangliosides, also occurs (Vanier, 2010, OrphanetJournal of Rare Diseases, vol. 5: 16). Owing to different clinicalpresentations and course of disease, NPC1 disease is typicallycategorized as early-infantile onset (<2 yrs), late-infantile onset (2to <6 years), juvenile onset (6 to <15 years), and adolescent/adultonset (>15 years).

Efforts to treat NPC in humans have focused on substrate reductiontherapy, such as inhibiting glycosphingolipid synthesis, for examplewith N-butyldeoxynojirimycin (miglustat, Zavesca®), or on amelioratingoverall lipid storage, particularly storage of cholesterol andglycosphingolipids, through clearance mechanisms.

2-Hydroxypropyl-beta-cyclodextrins have been shown to alleviate excesscholesterol storage in NPC cells (Abi-Mosleh, L. et al., Proceedings ofthe National Academy of Sciences USA, 2009, vol. 106 (46), pages19316-19321), consistent with a previous report of related cyclodextrinsextracting cholesterol from the plasma membrane of cells (Rodal, S. K.et al., 1999, Molecular Biology of the Cell, vol. 10, pages 961-974).Hydroxypropyl beta-cyclodextrins have also been observed to havebeneficial effects in animal models of NPC. For example, a compositioncomprising 2-hydroxypropyl-beta-cyclodextrins was reported to reversedefective lysosomal transport of cholesterol in the liver and brains ofNpc1 knockout mice, and led to a prolongation of life in these mutantscompared with no treatment (Liu, B. et al., 2009, Proceedings of theNational Academy of Sciences USA, vol. 106 (7), pages 2377-2382;Davidson et al., 2009, PLoS One 4:e6951).

Various hydroxypropyl beta-cyclodextrin compositions have beenadministered to human NPC patients in the United States, Brazil, andJapan under compassionate use exemptions, with anecdotal reports of someimprovement in various signs and symptoms. However, blinded clinicaltrials of hydroxypropyl beta-cyclodextrin compositions to determinesafety and efficacy have not been completed (Ottinger, E. A. et al.,2014, Current Topics in Medicinal Chemistry, vol. 14 (3), pages330-339). Given likely observer bias in the anecdotal reports, there isa need for controlled clinical studies to confirm that hydroxypropylbeta-cyclodextrin provides clinical benefit.

Effective treatment of NPC will require chronic intrathecal orintracerebroventricular administration beginning in infancy, andparenteral grade compositions of hydroxypropyl beta-cyclodextrins usedpreviously in human patients contain impurities that make themunsuitable for chronic administration directly to the cerebrospinalfluid of infants and children: propylene glycol, which is thought to beototoxic; beta-cyclodextrin molecules having no hydroxypropylsubstitutions, which are known to form precipitates and to have an acutetoxicity (Muller and Brauns, 1985, International Journal ofPharmaceutics, vol. 26, pages 77-88); and bacterial endotoxin, which ishighly inflammatory. There is, therefore, a need for pharmaceuticalcompositions of hydroxypropyl beta-cyclodextrins of higher purity.

In addition, all existing parenteral grade compositions of hydroxypropylbeta-cyclodextrins contain complex mixtures of hydroxypropylbeta-cyclodextrin species having different degrees of hydroxypropylsubstitution. The ratios of these species within the mixture differwidely among the various suppliers, and vary even among batches from asingle supplier. It is not known how these different species contributeto the pharmacological effects of the complex mixture. There is,therefore, a need for pharmaceutical compositions of hydroxypropylbeta-cyclodextrins having more precisely defined and preciselycontrolled mixtures, or fingerprints, of such species.

There is, finally, a need for methods of manufacturing at commercialscale under GMP conditions pharmaceutical compositions of hydroxypropylbeta-cyclodextrins suitable for chronic intrathecal orintracerebroventricular administration, having low levels of impurities,and having specific and structurally-defined composition.

SUMMARY

We analyzed initial data from a phase I clinical trial in which patientswith NPC type 1 disease are being treated by intrathecal administrationof 2-hydroxypropyl beta-cyclodextrin (“HPBCD”) using an existingparenteral grade composition, Kleptose® HPB (Roquette). In thisnon-randomized, open-label, single-center study conducted by the NIH,Kleptose® HPB is being administered via lumbar injection to drug-naivecohorts of patients at escalating doses. In certain of our analyses, wealso included data from three patients being treated with intrathecalKleptose® HPB at another institution under individual INDs.

Our analyses confirmed that intrathecal administration of Kleptose® HPBprovides therapeutic benefit in NPC type 1 disease. Using a standardaggregate outcome measure, the NPC Clinical Severity Scale, 7/15patients were observed to have stable or improving disease, as comparedto 0/13 in a cohort of patients in whom the natural history of untreateddisease has been studied. Using a new composite endpoint informed bypost-hoc analysis of the data, we found that 11/15 study patients showedstable or improving disease versus only 4/13 with stable disease in theNatural History cohort.

More detailed analyses, however, showed that while intrathecaladministration of HPBCD improves certain signs and symptoms of NPC type1 disease, it merely slows progression of others, and paradoxicallyappears to accelerate progression in other symptoms. In particular,hearing loss appears to have been accelerated in patients receivingintrathecal Kleptose® HPB. Our analysis of representative batches ofKleptose® HPB revealed that this parenteral grade product comprises acomplex mixture of beta-cyclodextrin molecules having different degreesof substitution; it is not known which of these species contributes tothe observed improvement, the slowing of progression, and theacceleration in progression of the various clinical domains.

To prepare for clinical trials in which HPBCD will be administereddirectly to the cerebrospinal fluid for longer periods of time, and withmore frequent dosing, we developed methods to reduce levels of propyleneglycol, which is a presumed ototoxin; beta-cyclodextrin molecules havingno hydroxypropyl substitutions, which are known to form precipitates;and bacterial endotoxin, which is highly inflammatory. Although themethods were successful in reducing the specified impurities, weobserved that absorption chromatography with alumina, whether used aloneor in combination with solvent precipitation, also changed thecompositional fingerprint, substantially reducing the amount ofbeta-cyclodextrin molecules having a single hydroxypropyl substitution(DS-1) and reducing the amount of beta-cyclodextrin molecules having twohydroxypropyl groups (DS-2). Reduction in the prevalence of moleculeswith low degrees of substitution (DS-0, as intended; and DS-1 and DS-2,unintended) increased the average degree of substitution (DS_(a)) of themixture.

Despite the change in fingerprint from Kleptose® HPB there was,surprisingly, no change in the expression of genes known to be involvedin cholesterol metabolism and transport, as assessed by in vitro geneexpression profiling experiments. This discovery will allow the morehighly purified and compositionally distinct HPBCD composition to beadministered by intrathecal or intracerebroventricular route to the CSFof patients with NPC disease for longer periods, optionally with morefrequent dosing, with therapeutic effect and improved safety.

Accordingly, in a first aspect, mixtures of beta-cyclodextrin moleculessubstituted at one or more hydroxyl positions by hydroxypropyl groups,the mixture optionally including unsubstituted beta-cyclodextrinmolecules, are provided. The mixture comprises less than 1%unsubstituted beta-cyclodextrin (“DS-0”) and beta-cyclodextrinsubstituted with one hydroxypropyl group (“DS-1”), collectively; atleast 85% beta-cyclodextrin substituted with three hydroxypropyl groups(“DS-3”), beta-cyclodextrin substituted with four hydroxypropyl groups(“DS-4”), beta-cyclodextrin substituted with five hydroxypropyl groups(“DS-5”), and beta-cyclodextrin substituted with six hydroxypropylgroups (“DS-6”), collectively; and less than 1% beta-cyclodextrinsubstituted with nine hydroxypropyl groups (“DS-9”) andbeta-cyclodextrin substituted with ten hydroxypropyl groups (“DS-10”),collectively, each as determined by peak height of an electrospray MSspectrum.

In certain embodiments, less than 0.1% of the beta-cyclodextrin mixtureis DS-0 and DS-1, collectively. In some embodiments, less than 0.01% ofthe beta-cyclodextrin mixture is DS-0 and DS-1, collectively. In someembodiments, at least 87% of the beta-cyclodextrin mixture is DS-3,DS-4, DS-5, and DS-6, collectively. In some embodiments, at least 90% ofthe beta-cyclodextrin mixture is DS-3, DS-4, DS-5, and DS-6,collectively. In some embodiments, less than 0.1% of thebeta-cyclodextrin mixture is DS-9 and DS-10, collectively. In certainembodiments, less than 0.01% of the beta-cyclodextrin mixture is DS-9and DS-10, collectively.

In another aspect, the mixture comprises less than 1% unsubstitutedbeta-cyclodextrin (“DS-0”) and beta-cyclodextrin substituted with onehydroxypropyl group (“DS-1”), collectively, and less than 1%beta-cyclodextrin substituted with nine hydroxypropyl groups (“DS-9”)and beta-cyclodextrin substituted with ten hydroxypropyl groups(“DS-10”), collectively, each as determined by peak height of anelectrospray MS spectrum, and the mixture has an average molarsubstitution (“MS”) in the range of 0.50 to 0.80.

In certain embodiments, less than 0.1% of the beta-cyclodextrin mixtureis DS-0 and DS-1, collectively. In some embodiments, less than 0.01% ofthe beta-cyclodextrin mixture is DS-0 and DS-1, collectively. In someembodiments, less than 0.1% of the beta-cyclodextrin mixture is DS-9 andDS-10, collectively. In certain embodiments, less than 0.01% of thebeta-cyclodextrin mixture is DS-9 and DS-10, collectively. In variousembodiments, the MS is in the range of 0.60 to 0.70. In some of theseembodiments, the MS is in the range of 0.64 to 0.68. In certainembodiments, the MS is about 0.66-0.67.

In another aspect, pharmaceutical compositions are provided, thepharmaceutical compositions comprising the beta-cyclodextrin mixturedescribed herein and a pharmaceutically acceptable diluent.

In some embodiments, the composition comprises no more than 0.5%propylene glycol, as measured by the HPLC method set forth in the USPHydroxypropyl Betadex monograph. In some embodiments, the compositioncomprises no more than 0.01% propylene glycol, as measured by the HPLCmethod set forth in the USP Hydroxypropyl Betadex monograph. In someembodiments, the pharmaceutical composition comprises no more than(“NMT”) 5 EU of endotoxins per gram of beta-cyclodextrin mixture. Inspecific embodiments, the pharmaceutical composition comprises NMT 1.5EU of endotoxins per gram of beta-cyclodextrin mixture. In someembodiments, the pharmaceutical composition comprises no more than 1 ppmpropylene oxide, determined according to the USP Hydroxypropyl Betadexmonograph.

In typical embodiments, the pharmaceutical composition is suitable forintrathecal or intracerebroventicular administration. In someembodiments, the pharmaceutical composition has an osmolality of about300 to about 450 mOsm/kg. In some embodiments, the composition comprisesabout 10 mg/mL to about 200 mg/mL of the beta-cyclodextrin mixture.

In another aspect, the pharmaceutical composition comprises a mixture ofbeta-cyclodextrin molecules substituted at one or more hydroxylpositions by hydroxypropyl groups, the mixture optionally includingunsubstituted beta-cyclodextrin molecules, and a diluent that ispharmaceutically acceptable for intrathecal, intracerebroventricular, orintravenous administration. The composition comprises no more than(“NMT”) 5 EU of endotoxins per gram of beta-cyclodextrin mixture, nomore than 0.5% propylene glycol, as measured by the HPLC method setforth in the USP Hydroxypropyl Betadex monograph, and no more than 1 ppmpropylene oxide, determined according to the USP Hydroxypropyl Betadexmonograph.

In some embodiments, the composition comprises NMT 1.5 EU of endotoxinsper gram of beta-cyclodextrin mixture. In some embodiments, thecomposition comprises no more than 0.01% propylene glycol, as measuredby the HPLC method set forth in the USP Hydroxypropyl Betadex monograph.In certain embodiments, the mixture comprises less than 3% unsubstitutedbeta-cyclodextrin (“DS-0”), beta-cyclodextrin substituted with onehydroxypropyl group (“DS-1”), and beta-cyclodextrin substituted with twohydroxypropyl groups (“DS-2”), collectively; at least 65%beta-cyclodextrin substituted with five hydroxypropyl groups (“DS-5”),beta-cyclodextrin substituted with six hydroxypropyl groups (DS-6″), andbeta-cyclodextrin substituted with seven hydroxypropyl groups (“DS-7”),collectively; and less than 3% beta-cyclodextrin substituted with ninehydroxypropyl groups (“DS-9”) and beta-cyclodextrin substituted with tenhydroxypropyl groups (“DS-10”), collectively, as determined by peakheights of an electrospray MS spectrum.

In another aspect, methods of treating Niemann-Pick disease Type C areprovided, the methods comprising administering to a patient in needthereof a therapeutically effective amount of the pharmaceuticalcomposition.

In typical embodiments, the composition is administered intrathecally orby intracerebroventricular administration. In some embodiments, themethod comprises administering about 300 mg to about 2000 mg of thebeta-cyclodextrin mixture to the patient. In certain embodiments, thecomposition is administered once every week, once every two weeks, onceevery three weeks, once every month, once every two months, or onceevery three months. In certain embodiments, the method comprisesadministering about 900 mg to about 1800 mg of the beta-cyclodextrinmixture to the patient once every two weeks. In certain embodiments, themethod comprises administering about 900 mg of the beta-cyclodextrinmixture to the patient once every two weeks.

In some embodiments, the method comprises administering an amount of thebeta-cyclodextrin mixture sufficient to modulate the level incerebrospinal fluid of one or more of: tau protein, amyloid peptide,neurofilament light protein (NFL), glial fibrillary acidic protein(GFAP), sterol, oxysterol, chitotriosidase activity, calbindin,lysosomal-associated membrane protein 1 (LAMP-1), GM2 or GM3ganglioside, sphingosine, and sphingosine-1-phosphate (SIP).

In some embodiments, the method comprises administering an amount of thebeta-cyclodextrin mixture sufficient to modulate the level in plasma ofone or more of: 7-ketocholesterol, 7β-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol,and cholestane-3β,5α,6β-triol.

In some embodiments, the method comprises administering an amount of thebeta-cyclodextrin mixture sufficient to modulate the level in urine ofone or more of 3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid(SNAG-Δ⁵-CA), glycine-conjugated3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CG),and taurine-conjugated3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CT).

In some embodiments, the method comprises administering thebeta-cyclodextrin mixture in an amount sufficient to maintain or reduceone or more domain scores of the NPC Severity Scale selected from:ambulation, fine motor skills, cognition, speech, swallowing, eyemovement, memory, hearing, and seizures.

In another aspect, a process for preparing the beta-cyclodextrin mixtureis presented, comprising treating Kleptose® HBP with absorptionchromatography on alumina.

In some embodiments, the process comprises a combination of absorptionchromatography on alumina and solvent precipitation. In someembodiments, the solvent precipitation is performed using water withacetone as precipitating agent. In other embodiments, the solventprecipitation is performed using methanol with acetone as precipitatingagent.

In another aspect, mixtures of beta-cyclodextrin molecules substitutedat one or more hydroxyl positions by hydroxypropyl groups, the mixtureoptionally including unsubstituted beta-cyclodextrin molecules, made bytreating Kleptose® HBP with a combination of absorption chromatographyon alumina and solvent precipitation.

In a further aspect, methods are provided for qualifying a mixture ofbeta-cyclodextrin molecules substituted at one or more hydroxylpositions by hydroxypropyl groups, the mixture optionally includingunsubstituted beta-cyclodextrin molecules, for use in a pharmaceuticalcomposition for intrathecal or intracerebroventricular administration.The method comprises (a) performing electrospray MS analysis of themixture; (b) measuring peak heights; and (c) calculating the percentageof each beta-cyclodextrin species in the entire mixture based on peakheights. The mixture is qualified for use—that is, is of qualitysufficient for use—if wherein the mixture comprises less than 1% DS-0and DS-1, collectively; at least 85% DS-3, DS-4, DS-5, and DS-6,collectively; and less than 1% DS-9 and DS-10, collectively.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising, as a pharmaceutically active ingredient, amixture of unsubstituted beta-cyclodextrin molecules andbeta-cyclodextrin molecules substituted at one or more hydroxylpositions by hydroxypropyl groups, wherein the mixture has an averagenumber of hydroxypropyl groups per beta-cyclodextrin (“DSa”) of about 3to about 7. In preferred embodiments, the pharmaceutical compositioncomprises no more than 0.5% propylene glycol, no more than (“NMT”) 1.5EU of endotoxin per gram of beta-cyclodextrin mixture, and no more than1% of the mixture is unsubstituted with a hydroxypropyl group (“DS-0”).In various preferred embodiments, the beta-cyclodextrin mixture has anaverage molar substitution (MS) in the range of about 0.58-about 0.68(DSa in the range of about 4.06-4.76). In certain of these preferredembodiments, the mixture has an MS of 0.58-0.68 (DSa of 4.06-4.76), andin some embodiment an MS of about 0.63. In various preferredembodiments, the mixture has an average molar substitution (MS) in therange of about 0.65 to about 0.68 (DSa 4.6-4.7), and in someembodiments, an average molar substitution of about 0.67.

In some embodiments, the beta-cyclodextrins in the mixture consist ofglucose units of the structure:

wherein R¹, R², and R³, independently for each occurrence, are —H or—HP, wherein HP comprises one or more hydroxypropyl groups.

In some embodiments, the average number of occurrences of HP perbeta-cyclodextrin is about 3 to about 7.

In some embodiments, at least 15% of total occurrences of R¹ and R²combined are HP.

In some embodiments, at least 30% of occurrences of R³ are HP.

In some embodiments, at least 70% of the beta-cyclodextrins collectivelyhave an average number of occurrences of HP per beta-cyclodextrin ofabout 4 to about 6.

In some embodiments, the DSa is about 3 to about 4. In some embodiments,the DSa is 3.3±0.3. In some embodiments, the DSa is 3.7±0.3.

In some embodiments, the DSa is about 3.5 to about 4.5. In someembodiments, the DSa is 3.8±0.3. In some embodiments, the DSa is4.2±0.3.

In some embodiments, the DSa is about 4 to about 5. In some embodiments,the DSa is 4.3±0.3. In some embodiments, the DSa is 4.7±0.3.

In some embodiments, the DSa is about 4.5 to about 5.5. In someembodiments, the DSa is 4.8±0.3. In some embodiments, the DSa is5.2±0.3.

In some embodiments, the DSa is about 5 to about 6. In some embodiments,the DSa is 5.3±0.3. In some embodiments, the DSa is 5.7±0.3.

In some embodiments, the DSa is about 5.5 to about 6.5. In someembodiments, the DSa is 5.8±0.3. In some embodiments, the DSa is6.2±0.3.

In some embodiments, the DSa is about 6 to about 7. In some embodiments,the DSa is 6.3±0.3. In some embodiments, the DSa is 6.7±0.3.

In some embodiments, at least 70% of the beta-cyclodextrins have a DSwithin DSa f 1. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DSa f 1.

In some embodiments, the hydroxypropyl groups are substituted at thehydroxyl positions of the beta-cyclodextrins as hydroxypropyl chains ofthe structure [CH2CH(CH3)O]_(n)H, wherein n≥1 and the average number ofhydroxypropyl chains per beta-cyclodextrin is about 3 to about 7. Insome embodiments, at least 70% of the hydroxypropyl chains have n=1. Insome embodiments, less than 30% of the hydroxypropyl chains have n=2. Insome embodiments, less than 10% of the hydroxypropyl chains have n>2. Insome embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is about 4 to about 6. In some embodiments, at least70% of the beta-cyclodextrins collectively have an average number ofhydroxypropyl chains per beta-cyclodextrin of about 4 to about 6.

In some embodiments, the pharmaceutical composition disclosed hereincontains less than about 10 International Units (IU), such as less thanabout 6 IU, less than about 3 IU, or less than about 1.5 IU, ofendotoxins per gram of the pharmaceutically active ingredient. The levelof endotoxins is determined by Limulus amoebocyte lysate test.

In some embodiments, the pharmaceutically active ingredient containsless than about 2% by weight, such as less than about 1% by weightunsubstituted beta-cyclodextrin.

In some embodiments, the pharmaceutically active ingredient containsless than about 0.5% by weight, such as less than about 0.2% by weightpropylene glycol or propylene glycol oligomers.

In some embodiments, the pharmaceutically active ingredient containsless than about 1 ppm propylene oxide.

In some embodiments, the pharmaceutical composition comprises apharmaceutically active ingredient wherein a 20% (w/v) solution of thepharmaceutically active ingredient in 1 mL of distilled watersolubilizes at least 2 mg, such as at least 3 mg, at least 4 mg, or atleast 5 mg, unesterified cholesterol at room temperature when measuredby UV spectrometry after about 24 hours.

In some embodiments, the pharmaceutical composition exhibits a lowerototoxicity than Trappsol® Cyclo. In some embodiments, the ototoxicityis determined in vitro by toxicity in a House Ear Institute-organ ofCorti 1 (HEI-OC1) cell. In some embodiments, ototoxicity is determinedin vivo by a brainstem auditory evoked response (BAER) test in asubject, such as a mouse, a rat, a cat, a dog, or a human.

In some embodiments, the pharmaceutical composition is suitable forintrathecal or intracerebroventricular administration.

In some embodiments, the pharmaceutical composition has an osmolality ofabout 300 to about 450 mOsm/kg.

In some embodiments, the pharmaceutical composition comprises about 10mg/mL to about 200 mg/mL of the pharmaceutically active ingredient.

In some embodiments, the sole pharmaceutically active ingredient of thepharmaceutical composition is obtained by purifying Kleptose® HBP,Kleptose® HP, Trappsol® Cyclo, or Cavasol® W7 HP Pharma. In certainembodiments, the sole pharmaceutically active ingredient of thepharmaceutical composition is obtained by purifying Kleptose® HBP. Incertain embodiments, the sole pharmaceutically active ingredient ofpharmaceutical composition is obtained by purifying Trappsol® Cyclo. Insome embodiments, purifying comprises hydrophilic or hydrophobicinteraction or affinity purification and can involve chromatographicmethods, such as purification by HPLC or gel chromatography.

The disclosure also provides a method of treating Niemann-Pick diseaseType C, comprising administering to a subject in need thereof, e.g., byintrathecal or intracerebroventricular administration, a therapeuticallyeffective amount of a pharmaceutical composition as described herein. Insome embodiments, the method comprises administering about 300 to about3000 mg of the pharmaceutically active ingredient to the patient. Insome embodiments, the administering occurs every week, every two weeks,every three weeks, every month, every two months, or every three months.For example, the method can comprise administering about 600 to about1800 mg of the pharmaceutically active ingredient to the subject everytwo weeks.

In some embodiments, the method comprises administration of an amount ofthe pharmaceutically active ingredient sufficient to modulate the levelin cerebrospinal fluid of one or more of: tau protein, amyloid peptide,neurofilament light protein (NFL), glial fibrillary acidic protein(GFAP), sterol, oxysterol, chitotriosidase activity, calbindin,lysosomal-associated membrane protein 1 (LAMP-1), GM2 or GM3ganglioside, sphingosine, and sphingosine-1-phosphate (S1P).

In some embodiments, the method comprises administration of an amount ofthe pharmaceutically active ingredient sufficient to modulate the levelin plasma of one or more of: 7-ketocholesterol, 7-hydroxycholesterol,24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol,and cholestane-3β,5α,6β-triol.

In some embodiments, the method comprises administration of an amount ofthe pharmaceutically active ingredient sufficient to modulate the levelin urine of one or more of:3-sulfoxy-7-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CA),glycine-conjugated 3-sulfoxy-7-N-acetylglucosaminyl-5-cholen-24-oic acid(SNAG-Δ⁵-CG), and taurine-conjugated3-sulfoxy-7-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CT).

In some embodiments, the method further comprises maintaining orreducing one or more domain scores of NPC Severity Scale selected from:ambulation, fine motor skills, cognition, speech, swallowing, eyemovement, memory, hearing, and seizures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the clinical domains contributing to the standard NPCClinical Severity Scale according to Yanjanin et al., “Linear ClinicalProgression, Independent of Age of Onset, in Niemann-Pick Disease, TypeC,” Am. J. Med. Genet. Part B 153B:132-140 (2010).

FIG. 2 summarizes the results from initial analyses of the Phase Iclinical trial described in Example 1.

FIG. 3 summarizes further analyses of the Phase I clinical trialdescribed in Example 1.

FIG. 4 summarizes analyses of the Phase I clinical trial data usingchange from baseline.

FIG. 5 summarizes early results from the Phase I clinical trial usingoverall NPC Scores.

FIG. 6 summarizes early results from the Phase I clinical trial withhearing impact removed.

FIG. 7 summarizes the clinical domains contributing to a novel “NPCComposite” Endpoint, or severity score;

FIG. 8 summarizes early results from the Phase I clinical trial using anovel NPC Composite Endpoint.

FIG. 9 depicts a representative HPLC chromatogram using a CD-screenmethod.

FIG. 10 depicts the comparative HPLC chromatograms using differentsolvents in a CD-screen method. Upper trace: methanol; lower trace:acetonitrile.

FIG. 11 depicts the mass spectrometry extracted ion chromatograms ofhydroxypropyl beta-cyclodextrins having a different degree ofsubstitution (DS) using a CD-screen column.

FIG. 12 depicts a representative HPLC chromatogram using a LiChrosphereC18 reverse phase column.

FIG. 13 depicts the comparative HPLC chromatograms using differentsolvents in a LiChrosphere C18 reverse phase chromatography method.Upper trace: methanol; lower trace: acetonitrile.

FIG. 14 depicts the mass spectrometry extracted ion chromatograms ofhydroxypropyl beta-cyclodextrins having a different degree ofsubstitution (DS) using the LiChrosphere C18 column.

FIG. 15 depicts the comparative HPLC chromatograms using differentsolvents in a HILIC column Upper trace: 79% acetonitrile in watergradient; lower trace: 75% acetonitrile.

FIG. 16 depicts the mass spectrometry extracted ion chromatograms ofhydroxypropyl beta-cyclodextrins having a different degree ofsubstitution (DS) using a HILIC column.

FIG. 17 depicts a representative HPLC chromatogram using a silica gelcolumn.

FIG. 18 depicts the mass spectrometry extracted ion chromatograms ofhydroxypropyl beta-cyclodextrins having a different degree ofsubstitution (DS) using a silica gel column.

FIG. 19 shows overlay HPLC traces for the commercial Kleptose HPB®(Roquette, batch E0223) (upper trace), and after purification bycomplexation with D-limonene (lower trace). X-axis shows retention timein minutes.

FIG. 20 shows HPLC traces for a sample obtained after solventprecipitation using water/acetone mixture (upper trace) in comparison tothe commercial Kleptose HPB® (Roquette, batch E0223).

FIG. 21 shows overlay HPLC traces for the commercial Kleptose HPB®(Roquette, batch E0223) (upper trace), and after purification by resinand charcoal treatment (lower trace). X-axis shows retention time inminutes.

FIG. 22 shows overlay HPLC traces for the commercial Kleptose HPB®(Roquette, batch E0223) (upper trace), and after purification by aluminaclarification. Lower trace shows data for the purified filtrate afteralumina clarification; middle trace shows data for the purified 1^(st)rinsing after alumina clarification. X-axis shows retention time inminutes.

FIG. 23 shows comparative HPLC traces for each of Fractions A through H(excluding E) obtained from alumina chromatography of Kleptose® HPB.Fractions A through H correspond to Samples 5.4.3.2.2A through5.4.3.2.2H, respectively (excluding E). X-axis shows retention time inminutes.

FIG. 24 shows a graph of the percentage impurity content in each ofFractions A through H collected from purification by aluminachromatography of Kleptose HPB®. A through H on the x-axis correspond toSamples 5.4.3.2.2A through 5.4.3.2.2H, respectively. Y-axis showspercent impurity content. PG %=percent propylene glycol (diagonal linedbars); total other impurities %=percent of total cyclodextrin-relatedimpurities (black bars); BCD %=percent unsubstituted beta-cyclodextrin(hollow bars); HPBCD DS-1%=percent of monosubstituted beta-cyclodextrins(reverse diagonal lined bars).

FIG. 25 depicts an exemplary gas chromatogram of Kleptose HPB®identifying propylene glycol (impurity to be measured) and ethyleneglycol (internal standard). X-axis shows time in minutes; y-axis showsresponse.

FIG. 26 depicts exemplary gas chromatograms of propylene glycolderivatives. X-axis shows time in minutes; y-axis shows response inmillivolts.

FIG. 27 depicts the calibration graph to determine propylene glycolconcentrations in gas chromatography samples. PG/EG=ratio of propyleneglycol to ethylene glycol as indicated.

FIG. 28 depicts an exemplary ¹H NMR spectrum of Kleptose HPB® (DS_(a) of4.1) using the European Pharmacopeial method.

FIG. 29A and FIG. 29B present electrospray MS spectra data from a firstlaboratory, with FIG. 29A showing the Kleptose® HPB spectrum and FIG.29B showing the spectrum of Trappsol® Cyclo™. Numbers have been added tothe spectra to identify the number of hydroxypropyl moieties in eachpeak.

FIG. 30A and FIG. 30B IG. Present electrospray MS data from a secondlaboratory, with FIG. 30A showing the spectrum of Kleptose® HPB and FIG.30B showing the spectrum of Trappsol® Cyclo™.

FIG. 31A, FIG. 31B, and FIG. 31C compare electrospray MS data from threedifferent lots of Kleptose® HPB, performed by two different labs.

FIG. 32A and FIG. 32B present electrospray MS spectra from two differentlots of Trappsol® Cyclo™, by two different laboratories, using the sameconditions as were used to generate the Kleptose data shown in FIG. 31A,FIG. 31B, and FIG. 31C.

FIG. 33A and FIG. 33B show electrospray MS spectra in which the Y axishas been expanded as compared to FIG. 29A, FIG. 29B, FIG. 30A, FIG. 30B,FIG. 31A, FIG. 31B, FIG. 31C, FIG. 32A, and FIG. 32B to show peaksbetween 1090 and 1230 m/z. FIG. 33A is the spectrum obtained fromTrappsol® Cyclo™. FIG. 33B is the spectrum obtained from Kleptose® HP.

FIG. 34A and FIG. 34B present electrospray MS data further comparing thedifferences between Kleptose® HPB and Trappsol® Cyclo™, with FIG. 34Ashowing the Kleptose® HPB spectrum and FIG. 34B showing the spectrum ofTrappsol® Cyclo™. Numbers have been added to the spectra to identify thenumber of hydroxypropyl moieties in each peak.

FIG. 35A and FIG. 35B present additional MS spectra differences betweenKleptose® HPB and Trappsol® Cyclo™ between 995 and 1095 m/z, with FIG.35A showing the Kleptose® HPB spectrum and FIG. 35B showing the spectrumof Trappsol® Cyclo™.

FIG. 36A and FIG. 36B present electrospray MS data showing the effect ofpurification with alumina adsorption on the substitution fingerprint.FIG. 36A shows the spectrum from the Kleptose® HPB starting material andFIG. 36B presents the spectrum from Batch CYL-4063, which was purifiedby combination of absorption chromatography on alumina and solventprecipitation (water-acetone).

FIG. 37 shows fold changes in expression of selected cholesterolhomeostasis-related genes in GM18453 and GM05659 cells treated with arange of Kleptose® HPB concentrations (0.1 mM to 10 mM).

FIG. 38 shows fold changes in expression in GM18453 cells, which arehomozygous for the NPC1 mutation, of the subset of cholesterolhomeostasis genes that in which expression was statisticallysignificantly different (p<0.001) upon treatment, for four differentcompositions: STD (Kleptose® HPB “standard”); AC (Kleptose® HPB purifiedby alumina chromatography); SP (Kleptose® HPB purified by solventprecipitation); and AP (Kleptose® HPB purified by alumina chromatography& solvent precipitation).

FIG. 39 shows the biological pathways that are most significantlyaffected, ranked by statistical significance, when GM18453 cells arerespectively treated with Kleptose® HPB and with a batch of Kleptose®HPB purified by a process that includes adsorption to aluminum.

FIG. 40 shows chromatograms of various fractions obtained frompreparative CD-Screen chromatographic separation of a batch of Kleptose®HPB, annotated to show the degree of substitution of thechromatographically separated hydroxypropyl beta-cyclodextrin species.

FIG. 41A, FIG. 41B, FIG. 41C, and FIG. 41D show electrospray MS spectraof Kleptose® HPB, the “L” fraction, the “M” fraction, and the “H”fraction, annotated to identify the signals by degree of hydroxypropylsubstitution, with FIG. 41A showing Kleptose® HPB batch E0245; FIG. 41Bshowing the “L” fraction (Fraction 2 alone); FIG. 41C showing the “M”fraction (pool of Fractions 4-15); and FIG. 41D showing the “H” fraction(pool of Fractions 16-24).

FIG. 42 shows the 10 biological pathways most affected by treatment ofthe NPC cells with 1.0 mM of the “L”, “M”, and “H” fractions, ranked indescending order of statistical significance.

FIG. 43 shows chromatograms of various fractions obtained frompreparative aluminum adsorption chromatographic separation of a batch ofKleptose® HPB (batch E0245), annotated to show the numerical fractionspooled to produce fractions “A”-“F” and “K”, and annotated to show thedegree of substitution of the chromatographically separatedhydroxypropyl beta-cyclodextrin species.

FIG. 44A and FIG. 44B show chromatograms of HPBCD mixture afterdifferent methods of purification, with FIG. 44A showing thechromatograms of purified HPBCD mixture after Methods II-IX, and FIG.44B showing the chromatograms of purified HPBCD mixture after Methods Xand XI.

FIG. 45 summarizes the analyses conducted for the NPC phase I clinicaltrial data at 18 months.

FIG. 46 shows the annualized rate of change of the Phase I clinicaltrial data at 18 months.

FIG. 47 shows the mean change from the baseline of the Phase I clinicaltrial data at 18 months.

FIG. 48 shows the responder analysis of the Phase I clinical trial dataat 18 months.

FIG. 49 shows the impact of treatment on hearing.

FIG. 50 summarizes the impact of treatment on hearing.

DETAILED DESCRIPTION

6.1. Experimental Observations

As described in detail below in Example 1, we analyzed initial data froma phase I clinical trial being conducted by the NIH in which patientswith NPC type 1 disease are being treated by intrathecal administrationof 2-hydroxypropyl beta-cyclodextrin (“HPBCD”) using an existingparenteral grade composition, Kleptose® HPB (Roquette). In thisnon-randomized, open-label, single-center study, Kleptose® HPB is beingadministered via lumbar injection to drug-naive cohorts of patients atescalating doses. In certain of our analyses, we also included data fromthree patients being treated with intrathecal Kleptose® HPB at anotherinstitution under individual INDs.

Our analyses confirmed that intrathecal administration of Kleptose® HPBprovides therapeutic benefit in NPC type 1 disease. Using a standardaggregate outcome measure, the NPC Clinical Severity Scale (see Yanjaninet al., “Linear Clinical Progression, Independent of Age of Onset, inNiemann-Pick Disease, Type C,” Am. J. Med. Genet. Part B 153B:132-140(2010); see also FIG. 1 and Table 1 herein), 7/15 patients were observedto have stable or improving disease, as compared to 0/13 in a cohort ofpatients in whom the natural history of untreated disease has beenstudied (see FIG. 5). Using a new composite endpoint informed bypost-hoc analysis of the data (FIG. 7), we found that 11/15 studypatients showed stable or improving disease versus only 4/13 with stabledisease in the Natural History cohort (see FIG. 8). In certain of ouranalyses, we used the NPC Clinical Severity Score with hearing andauditory brainstem response (ABR) removed.

More detailed analyses, however, showed that while intrathecaladministration of HPBCD improves certain signs and symptoms of NPC type1 disease, it merely slows progression of others, and paradoxicallyappears to accelerate progression in other symptoms. In particular,hearing loss appears to have been accelerated in patients receivingintrathecal Kleptose® HPB (see, e.g., FIGS. 2, 3, 4).

As set forth below in detail in Example 3, we analyzed representativebatches of Kleptose® HPB by various chromatographic methods. Theseanalyses revealed that this parenteral grade product comprises a complexmixture of beta-cyclodextrin molecules having different degrees ofsubstitution (see, e.g., FIGS. 11, 14, 16, 18); it is not known which ofthese species contributes to the observed improvement, the slowing ofprogression, and the acceleration in progression of the various clinicaldomains.

Further analyses using electrospray mass spectrometry, described inExample 5, demonstrated that there are significant differences in thesubstitution fingerprint of the hydroxypropyl beta-cyclodextrincomposition used in the phase I clinical trial described in Example 1,Kleptose® HPB, as compared to the substitution fingerprint of adifferent commercially available hydroxypropyl beta-cyclodextrincomposition, Trappsol® Cyclo™. We found that Kleptose® HPB has lowlot-to-lot variability in the substitution fingerprint, and low levelsof impurities, notably propylene glycol. In contrast, we found thatTrappsol® Cyclo™ exhibits high lot-to-lot variability in itssubstitution fingerprint and significantly higher levels of propyleneglycol, a presumed ototoxin.

To prepare for clinical trials in which HPBCD will be administereddirectly to the cerebrospinal fluid for longer periods of time, andpossibly with more frequent dosing, we developed methods to reducelevels of propylene glycol, which is a presumed ototoxin;beta-cyclodextrin molecules having no hydroxypropyl substitutions, whichare known to form precipitates; and bacterial endotoxin, which is highlyinflammatory, as described in Examples 6 and 7. Although the methodswere successful in reducing the specified impurities, we observed thatabsorption chromatography with alumina, whether used alone or incombination with solvent precipitation, also changed the compositionalfingerprint, substantially reducing the amount of beta-cyclodextrinmolecules having a single hydroxypropyl substitution (DS-1) and reducingthe amount of beta-cyclodextrin molecules having two substitutions(DS-2) (see Example 7; Table 20). Reduction in the prevalence ofmolecules with low degrees of substitution (DS-0, as intended; and DS-1and DS-2, unintended) increased the average degree of substitution(DS_(a)) of the mixture.

As detailed in Example 8 and summarized in FIGS. 35-38, gene expressionprofiling experiments using Kleptose® HPB and a batch of Kleptose® HPBfurther purified using adsorption to aluminum demonstrate that theactivity of the hydroxypropyl beta-cyclodextrin mixtures on cellshomozygous for the NPC1 mutation is a composite of the activitiesseparately contributed by species having different degrees ofhydroxypropyl substitution. Despite the change in compositionalfingerprint as compared to Kleptose® HPB, there was, surprisingly, nochange in the expression of genes known to be involved in cholesterolmetabolism and transport. This discovery will allow the novel, morehighly purified, and compositionally distinct HPBCD composition to beadministered by intrathecal or intracerebroventricular route to the CSFof patients with NPC disease for longer periods, with therapeutic effectand increased safety.

6.2. Pharmaceutical Compositions

The present disclosure provides a pharmaceutical composition comprising,as a pharmaceutically active ingredient, a mixture of beta-cyclodextrinmolecules substituted at one or more hydroxyl positions by hydroxypropylgroups, the mixture optionally including unsubstituted beta-cyclodextrinmolecules.

6.2.1. Pharmaceutically Active Ingredient

The pharmaceutically active ingredient is a mixture of beta-cyclodextrinmolecules substituted at one or more hydroxyl positions by hydroxypropylgroups, the mixture optionally including unsubstituted beta-cyclodextrinmolecules. The term “pharmaceutically active ingredient” is usedsynonymously with “active pharmaceutical ingredient” in this disclosure.

6.2.1.1. Average Degree of Substitution

As used herein, “substituted at one or more hydroxyl positions byhydroxypropyl groups” refers to replacement of the hydrogen of one ormore hydroxyl groups of a beta-cyclodextrin molecule with ahydroxypropyl group or a hydroxypropyl oligomer. For instance,“substituted at one or more hydroxyl positions by hydroxypropyl groups”can refer to an insertion of one or more —CH₂CH(CH₃)O— substituentswithin one or more O—H bonds on a beta-cyclodextrin molecule resultingin one or more ether linkages.

The number of hydroxypropyl groups per anhydroglucose unit in themixture of beta-cyclodextrins is the “molar substitution”, or “MS”, andis determined according to the procedures set forth in the USP monographon Hydroxypropyl Betadex (USP NF 2015) (“USP Hydroxypropyl Betadexmonograph”), incorporated herein by reference in its entirety. In thisdisclosure, the term “average molar substitution”, or “MS_(a)”, is usedsynonymously with “MS” as that term is used in the USP HydroxypropylBetadex monograph, and the term “glucose unit” is used as a synonym for“anhydroglucose unit” as that term is used in the USP HydroxypropylBetadex monograph.

As used herein, the “degree of substitution” or “DS” refers to the totalnumber of hydroxypropyl groups substituted directly or indirectly on abeta-cyclodextrin molecule. For example, a beta-cyclodextrin moleculecontaining glucose units, each of which is substituted with onehydroxypropyl group, has a DS=7. In another example, a beta-cyclodextrinmolecule in which only one of the seven glucose units is substitutedwith a hydroxypropyl group, and that hydroxypropyl group is itselfsubstituted with another hydroxypropyl group (e.g., a beta-cyclodextrinwith a single occurrence of HP that comprises two hydroxypropyl groups),has a DS=2.

As used herein, the “average number of hydroxypropyl groups perbeta-cyclodextrin,” also known as an “average degree of substitution,”“average DS,” or “DS_(a),” refers to the total number of hydroxypropylgroups in a population of beta-cyclodextrins divided by the number ofbeta-cyclodextrin molecules. In an illustrative example, an equal partsmixture of beta-cyclodextrins containing glucose units that are eachsubstituted with one hydroxypropyl group and beta-cyclodextrinscontaining glucose units that are each substituted with twohydroxypropyl groups has a DS_(a)=10.5 (average of equal partsbeta-cyclodextrins with DS=7 and DS=14). In another illustrativeexample, a mixture of 33.3% beta-cyclodextrins in which only one of theseven glucose units is substituted with a hydroxypropyl group (i.e.,DS=1) and 66.7% beta-cyclodextrins containing glucose units that areeach substituted with one hydroxypropyl group (i.e., DS=7) has aDS_(a)=5.0.

The DS_(a) is determined by multiplying the MS by 7. As used herein,DS_(a) is used synonymously with “degree of substitution” as that termis defined in the USP Hydroxypropyl Betadex monograph.

In some embodiments, the beta-cyclodextrins in the mixture consist ofglucose units of the structure:

wherein R¹, R², and R³, independently for each occurrence, are —H or—HP, wherein HP comprises one or more hydroxypropyl groups.

In some embodiments, HP comprises one hydroxypropyl group. In someembodiments, HP consists essentially of one hydroxypropyl group. In someembodiments, HP consists of one hydroxypropyl group.

In some embodiments, the average number of occurrences of HP perbeta-cyclodextrin is about 3 to about 7, e.g., about 3 to about 6, about3 to about 5, about 3 to about 4, about 4 to about 7, about 4 to about6, about 4 to about 5, about 5 to about 7, about 5 to about 6, or about6 to about 7.

In some embodiments, the total occurrences of R³=HP are greater than thetotal occurrences of either R¹=HP or R²=HP. In certain embodiments, thetotal occurrences of R³=HP are greater than the total combinedoccurrences of R¹=HP and R²=HP.

In some embodiments, at least about 5%, e.g., at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, or at least about 45% oftotal occurrences of R¹ and R² combined are HP.

In some embodiments, not more than about 95%, e.g., not more than about90%, not more than about 85%, not more than about 80%, not more thanabout 75%, not more than about 70%, not more than about 65%, not morethan about 60%, not more than about 55%, or not more than about 50% oftotal occurrences of R¹ and R² combined are HP.

In some embodiments, the percentage of R¹ and R² combined that are HPranges from about 5% to about 95%, such as about 10% to about 95%, about15% to about 95%, about 20% to about 95%, about 25% to about 95%, about30% to about 95%, about 35% to about 95%, about 40% to about 95%, about45% to about 95%, about 50% to about 95%, about 55% to about 95%, about60% to about 95%, about 65% to about 95%, about 70% to about 95%, about75% to about 95%, about 80% to about 95%, about 85% to about 95%, about90% to about 95%; such as from about 5% to about 90%, about 10% to about90%, about 15% to about 90%, about 20% to about 90%, about 25% to about90%, about 30% to about 90%, about 35% to about 90%, about 40% to about90%, about 45% to about 90%, about 50% to about 90%, about 55% to about90%, about 60% to about 90%, about 65% to about 90%, about 70% to about90%, about 75% to about 90%, about 80% to about 90%, about 85% to about90%; such as from about 5% to about 85%, about 10% to about 85%, about15% to about 85%, about 20% to about 85%, about 25% to about 85%, about30% to about 85%, about 35% to about 85%, about 40% to about 85%, about45% to about 85%, about 50% to about 85%, about 55% to about 85%, about60% to about 85%, about 65% to about 85%, about 70% to about 85%, about75% to about 85%, about 80% to about 85%; such as from about 5% to about80%, about 10% to about 80%, about 15% to about 80%, about 20% to about80%, about 25% to about 80%, about 30% to about 80%, about 35% to about80%, about 40% to about 80%, about 45% to about 80%, about 50% to about80%, about 55% to about 80%, about 60% to about 80%, about 65% to about80%, about 70% to about 80%, about 75% to about 80%; such as from about5% to about 75%, about 10% to about 75%, about 15% to about 75%, about20% to about 75%, about 25% to about 75%, about 30% to about 75%, about35% to about 75%, about 40% to about 75%, about 45% to about 75%, about50% to about 75%, about 55% to about 75%, about 60% to about 75%, about65% to about 75%, about 70% to about 75%; such as from about 5% to about70%, about 10% to about 70%, about 15% to about 70%, about 20% to about70%, about 25% to about 70%, about 30% to about 70%, about 35% to about70%, about 40% to about 70%, about 45% to about 70%, about 50% to about70%, about 55% to about 70%, about 60% to about 70%, about 65% to about70%; such as from about 5% to about 65%, about 10% to about 65%, about15% to about 65%, about 20% to about 65%, about 25% to about 65%, about30% to about 65%, about 35% to about 65%, about 40% to about 65%, about45% to about 65%, about 50% to about 65%, about 55% to about 65%, about60% to about 65%; such as from about 5% to about 60%, about 10% to about60%, about 15% to about 60%, about 20% to about 60%, about 25% to about60%, about 30% to about 60%, about 35% to about 60%, about 40% to about60%, about 45% to about 60%, about 50% to about 60%, about 55% to about60%; such as from about 5% to about 55%, about 10% to about 55%, about15% to about 55%, about 20% to about 55%, about 25% to about 55%, about30% to about 55%, about 35% to about 55%, about 40% to about 55%, about45% to about 55%, about 50% to about 55%; such as from about 5% to about50%, about 10% to about 50%, about 15% to about 50%, about 20% to about50%, about 25% to about 50%, about 30% to about 50%, about 35% to about50%, about 40% to about 50%, about 45% to about 50%; such as from about5% to about 45%, about 10% to about 45%, about 15% to about 45%, about20% to about 45%, about 25% to about 45%, about 30% to about 45%, about35% to about 45%, about 40% to about 45%; such as from about 5% to about40%, about 10% to about 40%, about 15% to about 40%, about 20% to about40%, about 25% to about 40%, about 30% to about 40%, about 35% to about40%; such as from about 5% to about 35%, about 10% to about 35%, about15% to about 35%, about 20% to about 35%, about 25% to about 35%, about30% to about 35%; such as from about 5% to about 30%, about 10% to about30%, about 15% to about 30%, about 20% to about 30%, about 25% to about30%; such as from about 5% to about 25%, about 10% to about 25%, about15% to about 25%, about 20% to about 25%; such as from about 5% to about20%, about 10% to about 20%, about 15% to about 20%; such as from about5% to about 15%, about 10% to about 15%; or about 5% to about 10%.

In some embodiments, at least about 5%, e.g., at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, or atleast about 50% of occurrences of R³ are HP.

In some embodiments, not more than about 95%, e.g., not more than about90%, not more than about 85%, not more than about 80%, not more thanabout 75%, not more than about 70%, not more than about 65%, not morethan about 60%, or not more than about 55% of occurrences of R³ are HP.

In some embodiments, the percentage of occurrence of R³ that are HPranges from about 20% to about 90%, e.g., about 25% to about 90%, about30% to about 90%, about 35% to about 90%, about 40% to about 90%, about45% to about 90%, about 50% to about 90%, about 55% to about 90%, about60% to about 90%, about 65% to about 90%, about 70% to about 90%, about75% to about 90%, about 80% to about 90%, about 85% to about 90%, about20% to about 85%, about 25% to about 85%, about 30% to about 85%, about35% to about 85%, about 40% to about 85%, about 45% to about 85%, about50% to about 85%, about 55% to about 85%, about 60% to about 85%, about65% to about 85%, about 70% to about 85%, about 75% to about 85%, about80% to about 85%, about 20% to about 80%, about 25% to about 80%, about30% to about 80%, about 35% to about 80%, about 40% to about 80%, about45% to about 80%, about 50% to about 80%, about 55% to about 80%, about60% to about 80%, about 65% to about 80%, about 70% to about 80%, about75% to about 80%, about 20% to about 75%, about 25% to about 75%, about30% to about 75%, about 35% to about 75%, about 40% to about 75%, about45% to about 75%, about 50% to about 75%, about 55% to about 75%, about60% to about 75%, about 65% to about 75%, about 70% to about 75%, about20% to about 70%, about 25% to about 70%, about 30% to about 70%, about35% to about 70%, about 40% to about 70%, about 45% to about 70%, about50% to about 70%, about 55% to about 70%, about 60% to about 70%, about65% to about 70%, about 20% to about 65%, about 25% to about 65%, about30% to about 65%, about 35% to about 65%, about 40% to about 65%, about45% to about 65%, about 50% to about 65%, about 55% to about 65%, about60% to about 65%, about 20% to about 60%, about 25% to about 60%, about30% to about 60%, about 35% to about 60%, about 40% to about 60%, about45% to about 60%, about 50% to about 60%, about 55% to about 60%, about20% to about 55%, about 25% to about 55%, about 30% to about 55%, about35% to about 55%, about 40% to about 55%, about 45% to about 55%, about50% to about 55%, about 20% to about 50%, about 25% to about 50%, about30% to about 50%, about 35% to about 50%, about 40% to about 50%, about45% to about 50%, about 20% to about 45%, about 25% to about 45%, about30% to about 45%, about 35% to about 45%, about 40% to about 45%, about5% to about 40%, about 10% to about 40%, about 15% to about 40%, about20% to about 40%, about 25% to about 40%, about 30% to about 40%, about35% to about 40%, about 20% to about 35%, about 25% to about 35%, about30% to about 35%, about 20% to about 30%, about 25% to about 30%, orabout 20% to about 25%.

In some embodiments, at least about 70%, e.g., at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at leastabout 95%, of the beta-cyclodextrins collectively have an average numberof occurrences of HP per beta-cyclodextrin of about 4 to about 7, e.g.,about 4 to about 6, about 4 to about 5, about 5 to about 7, about 5 toabout 6, or about 6 to about 7.

In some embodiments, the percentage of beta-cyclodextrins thatcollectively have an average number of occurrences of HP perbeta-cyclodextrin of about 4 to about 7, e.g., about 4 to about 6, about4 to about 5, about 5 to about 7, about 5 to about 6, or about 6 toabout 7, ranges from about 50% to about 99%, such as about 55% to about99%, about 60% to about 99%, about 65% to about 99%, about 70% to about99%, about 75% to about 99%, about 80% to about 99%, about 85% to about99%, about 90% to about 99%, about 95% to about 99%; such as from about50% to about 97%, such as about 55% to about 97%, about 60% to about97%, about 65% to about 97%, about 70% to about 97%, about 75% to about97%, about 80% to about 97%, about 85% to about 97%, about 90% to about97%, about 95% to about 97%; such as from about 50% to about 95%, about55% to about 95%, about 60% to about 95%, about 65% to about 95%, about70% to about 95%, about 75% to about 95%, about 80% to about 95%, about85% to about 95%, about 90% to about 95%; such as from about 50% toabout 90%, about 55% to about 90%, about 60% to about 90%, about 65% toabout 90%, about 70% to about 90%, about 75% to about 90%, about 80% toabout 90%, about 85% to about 90%; such as from about 50% to about 85%,about 55% to about 85%, about 60% to about 85%, about 65% to about 85%,about 70% to about 85%, about 75% to about 85%, about 80% to about 85%;such as from about 50% to about 80%, about 55% to about 80%, about 60%to about 80%, about 65% to about 80%, about 70% to about 80%, about 75%to about 80%; such as from about 50% to about 75%, about 55% to about75%, about 60% to about 75%, about 65% to about 75%, about 70% to about75%; such as from about 50% to about 70%, about 55% to about 70%, about60% to about 70%, about 65% to about 70%; such as from about 50% toabout 65%, about 55% to about 65%, about 60% to about 65%; such as fromabout 50% to about 60%, about 55% to about 60%; or such as from about50% to about 55%.

In certain embodiments, the pharmaceutical compositions of thedisclosure comprise, as a pharmaceutically active ingredient, a mixtureof unsubstituted beta-cyclodextrin molecules and beta-cyclodextrinmolecules substituted at one or more hydroxyl positions by hydroxypropylgroups, wherein the mixture has an average number of hydroxypropylgroups per beta-cyclodextrin molecule (DS_(a)) of about 3 to about 7.

In some embodiments, the DS_(a) is about 3 to about 5, such as about 3to about 4. In some embodiments, the DS_(a) is 3.3±0.3, 3.5±0.3, or3.7±0.3. In other embodiments, the DS_(a) is 3.2±0.2, 3.3±0.2, 3.4±0.2,3.5±0.2, 3.6±0.2, 3.7±0.2, or 3.8±0.2. In other embodiments, the DS_(a)is 3.1±0.1, 3.2±0.1, 3.3±0.1, 3.4±0.1, 3.5±0.1, 3.6±0.1, 3.7±0.1,3.8±0.1, or 3.9±0.1.

In some embodiments, the DS_(a) is about 3.5 to about 5.5, such as about3.5 to about 4.5. In some embodiments, the DS_(a) is 3.8±0.3, 4.0±0.3,or 4.2±0.3. In other embodiments, the DS_(a) is 3.7±0.2, 3.8±0.2,3.9±0.2, 4.0±0.2, 4.1±0.2, 4.2±0.2, or 4.3±0.2. In other embodiments,the DS_(a) is 3.6±0.1, 3.7±0.1, 3.8±0.1, 3.9±0.1, 4.0±0.1, 4.1±0.1,4.2±0.1, 4.3±0.1, or 4.4±0.1.

In some embodiments, the DS_(a) is about 4 to about 6, such as about 4to about 5. In some embodiments, the DS_(a) is 4.3±0.3, 4.5±0.3, or4.7±0.3. In other embodiments, the DS_(a) is 4.2±0.2, 4.3±0.2, 4.4±0.2,4.5±0.2, 4.6±0.2, 4.7±0.2, or 4.8±0.2. In other embodiments, the DS_(a)is 4.1±0.1, 4.2±0.1, 4.3±0.1, 4.4±0.1, 4.5±0.1, 4.6±0.1, 4.7±0.1,4.8±0.1, or 4.9±0.1.

In some embodiments, the DS_(a) is about 4.5 to about 6.5, such as about4.5 to about 5.5. In some embodiments, the DS_(a) is 4.8±0.3, 5.0±0.3,or 5.2±0.3. In other embodiments, the DS_(a) is 4.7±0.2, 4.8±0.2,4.9±0.2, 5.0±0.2, 5.1±0.2, 5.2±0.2, or 5.3±0.2. In other embodiments,the DS_(a) is 4.6±0.1, 4.7±0.1, 4.8±0.1, 4.9±0.1, 5.0±0.1, 5.1±0.1,5.2±0.1, 5.3±0.1, or 5.4±0.1.

In some embodiments, the DS_(a) is about 5 to about 7, such as about 5to about 6. In some embodiments, the DS_(a) is 5.3±0.3, 5.5±0.3, or5.7±0.3. In other embodiments, the DS_(a) is 5.2±0.2, 5.3±0.2, 5.4±0.2,5.5±0.2, 5.6±0.2, 5.7±0.2, or 5.8±0.2. In other embodiments, the DS_(a)is 5.1±0.1, 5.2±0.1, 5.3±0.1, 5.4±0.1, 5.5±0.1, 5.6±0.1, 5.7±0.1,5.8±0.1, or 5.9±0.1.

In some embodiments, the DS_(a) is about 5.5 to about 6.5. In someembodiments, the DS_(a) is 5.8±0.3, 6.0±0.3, or 6.2±0.3. In otherembodiments, the DS_(a) is 5.7±0.2, 5.8±0.2, 5.9±0.2, 6.0±0.2, 6.1±0.2,6.2±0.2, or 6.3±0.2. In other embodiments, the DS_(a) is 5.6±0.1,5.7±0.1, 5.8±0.1, 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, or6.4±0.1.

In some embodiments, the DS_(a) is about 6 to about 7. In someembodiments, the DS_(a) is 6.3±0.3, 6.5±0.3, or 6.7±0.3. In otherembodiments, the DS_(a) is 6.2±0.2, 6.3±0.2, 6.4±0.2, 6.5±0.2, 6.6±0.2,6.7±0.2, or 6.8±0.2. In other embodiments, the DS_(a) is 6.1±0.1,6.2±0.1, 6.3±0.1, 6.4±0.1, 6.5±0.1, 6.6±0.1, 6.7±0.1, 6.8±0.1, or6.9±0.1.

In some embodiments, the DS_(a) is about 4.1±15%, about 4.2±15%, about4.3±15%, about 4.4±15%, or about 4.5±15%, such as about 4.1±10%, about4.2±10%, about 4.3±10%, about 4.4±10%, or about 4.5±10%, such as about4.1±5%, about 4.2±5%, about 4.3±5%, about 4.4±5%, or about 4.5±5%. Forexample, in certain embodiments, the DS_(a) is about 4.31±10%, about4.32±10%, about 4.33±10%, about 4.34±10%, about 4.35±10%, about4.36±10%, or about 4.37±10%, such as about 4.31±5%, about 4.32±5%, about4.33±5%, about 4.34±5%, about 4.35±5%, about 4.36±5%, or about 4.37±5%.In particular embodiments, the DS_(a) is about 4.34±10%, such as about4.34±5%.

In some embodiments, the DS_(a) is about 4.3±15%, about 4.4±15%, about4.5±15%, about 4.6±15%, or about 4.7±15%, such as about 4.3±10%, about4.4±10%, about 4.5±10%, about 4.6±10%, or about 4.7±10%, such as about4.3±5%, about 4.4±5%, about 4.5±5%, about 4.6±5%, or about 4.7±5%. Forexample, in certain embodiments, the DS_(a) is about 4.47±10%, about4.48±10%, about 4.49±10%, about 4.50±10%, about 4.51±10%, about4.52±10%, or about 4.53±10%, such as about 4.47±5%, about 4.48±5%, about4.49±5%, about 4.50±5%, about 4.51±5%, about 4.52±5%, or about 4.53±5%.In particular embodiments, the DS_(a) is about 4.50±10%, such as about4.50±5%.

In some embodiments, the DS_(a) is about 6.1±15%, about 6.2±15%, about6.3±15%, about 6.4±15%, or about 6.5±15%, such as about 6.1±10%, about6.2±10%, about 6.3±10%, about 6.4±10%, or about 6.5±10%, such as about6.1±5%, about 6.2±5%, about 6.3±5%, about 6.4±5%, or about 6.5±5%. Forexample, in certain embodiments, the DS_(a) is about 6.34±10%, about6.35±10%, about 6.36±10%, about 6.37±10%, about 6.38±10%, about6.39±10%, or about 6.40±10%, such as about 6.34±5%, about 6.35±5%, about6.36±5%, about 6.37±5%, about 6.38±5%, about 6.39±5%, or about 6.40±5%.In particular embodiments, the DS_(a) is about 6.37±10%, such as about6.37±5%.

In some embodiments, the DS_(a) is about 6.3±15%, about 6.4±15%, about6.5±15%, about 6.6±15%, or about 6.7±15%, such as about 6.3±10%, about6.4±10%, about 6.5±10%, about 6.6±10%, or about 6.7±10%, such as about6.3±5%, about 6.4±5%, about 6.5±5%, about 6.6±5%, or about 6.7±5%. Forexample, in certain embodiments, the DS_(a) is about 6.50±10%, about6.51±10%, about 6.52±10%, about 6.53±10%, about 6.54±10%, about6.55±10%, or about 6.56±10%, such as about 6.50±5%, about 6.51±5%, about6.52±5%, about 6.53±5%, about 6.54±5%, about 6.55±5%, or about 6.56±5%.In particular embodiments, the DS_(a) is about 6.53±10%, such as about6.53±5%.

The distribution of the degree of substitution within a mixture ofunsubstituted beta-cyclodextrin molecules and beta-cyclodextrinmolecules substituted at one or more hydroxyl positions by hydroxypropylgroups can vary. For example, an equal parts mixture ofbeta-cyclodextrins containing glucose units each of which is substitutedwith one hydroxypropyl group and beta-cyclodextrins containing glucoseunits each of which is substituted with two hydroxypropyl groups has aDS_(a)=10.5 (average of equal parts beta-cyclodextrins with DS=7 andDS=14). Although DS_(a)=10.5, in this example there are nobeta-cyclodextrins having DS=10 or DS=11 within the mixture. In othercases, the majority of beta-cyclodextrins within the mixture ofbeta-cyclodextrins have DS that are close to the DS_(a).

In some embodiments of the disclosure, at least about 50%, e.g., atleast about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 97%, of the beta-cyclodextrinswithin the mixture have a DS within DS_(a)±Xσ, wherein σ is the standarddeviation, and X is 1, 2, or 3. For example, in some embodiments, atleast about 50%, e.g., at least about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about97%, of the beta-cyclodextrins within the mixture have a DS withinDS_(a)±1σ. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±1σ. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±1σ. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±1σ.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins within the mixturehave a DS within DS_(a)±2σ. In some embodiments, at least 70% of thebeta-cyclodextrins have a DS within DS_(a)±2σ. In some embodiments, atleast 90% of the beta-cyclodextrins have a DS within DS_(a)±2σ. In someembodiments, at least 95% of the beta-cyclodextrins have a DS withinDS_(a)±2σ.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins within the mixturehave a DS within DS_(a)±3σ. In some embodiments, at least 70% of thebeta-cyclodextrins have a DS within DS_(a)±3σ. In some embodiments, atleast 90% of the beta-cyclodextrins have a DS within DS_(a)±3σ. In someembodiments, at least 95% of the beta-cyclodextrins have a DS withinDS_(a)±3σ.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±1. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±1. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±1. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±1.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.8. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.8. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.8. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.8.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.6. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.6. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.6. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.6.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.5. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.5. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.5. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.5.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.4. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.4. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.4. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.4.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.3. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.3. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.3. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.3.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.2. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.2. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.2. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.2.

In some embodiments, at least about 50%, e.g., at least about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 97%, of the beta-cyclodextrins have a DS withinDS_(a)±0.1. In some embodiments, at least 70% of the beta-cyclodextrinshave a DS within DS_(a)±0.1. In some embodiments, at least 90% of thebeta-cyclodextrins have a DS within DS_(a)±0.1. In some embodiments, atleast 95% of the beta-cyclodextrins have a DS within DS_(a)±0.1.

In some embodiments, the MS ranges from 0.40 to 0.80, such as 0.41 to0.79, 0.42 to 0.78, 0.43 to 0.77, 0.44 to 0.76, 0.45 to 0.75, 0.46 to0.74, 0.47 to 0.73, 0.48 to 0.72, 0.49 to 0.71, 0.50 to 0.70, 0.51 to0.69, 0.52 to 0.68, 0.53 to 0.67, 0.54 to 0.66, 0.55 to 0.65, 0.56 to0.64, 0.57 to 0.63, 0.58 to 0.62, or 0.59 to 0.61.

In certain embodiments, the MS is about 0.40, about 0.41, about 0.42,about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48,about 0.49, about 0.50, about 0.51, about 0.52, about 0.53, about 0.54,about 0.55, about 0.56, about 0.57, about 0.58, about 0.59, about 0.60,about 0.61, about 0.62, about 0.63, about 0.64, about 0.65, about 0.66,about 0.67, about 0.68, about 0.69, about 0.70, about 0.71, about 0.72,about 0.73, about 0.74, about 0.75, about 0.76, about 0.77, about 0.78,about 0.79, or about 0.80.

In certain embodiments, the MS is about 0.571-0.686 (DS_(a) about 4.0 toabout 4.8). In some of these embodiments, the MS is in the range ofabout 0.58 to about 0.68. In currently preferred embodiments, the MS isin the range of 0.58-0.68.

In various embodiments, the MS is at least about 0.55. In certainembodiments, the MS is at least about 0.56, 0.57, 0.58, 0.59, or 0.60.In certain embodiments, the MS is no more than about 0.70. In specificembodiments, the MS is no more than about 0.69, 0.68, 0.67, 0.66, or0.65.

Hydroxypropyl groups can be bonded to the beta-cyclodextrins asmonomers, or can themselves be sequentially bonded to one or moreadditional hydroxypropyl groups to form hydroxypropyl oligomers whichare then bonded to the beta-cyclodextrins. In some embodiments, thehydroxypropyl groups are substituted at the hydroxyl positions of thebeta-cyclodextrins as hydroxypropyl chains of the structure—[CH₂CH(CH₃)O]_(n)H, wherein n≥1 and the average number of hydroxypropylchains per beta-cyclodextrin is about 3 to about 7, e.g., about 3 toabout 6, about 3 to about 5, about 3 to about 4, about 4 to about 7,about 4 to about 6, about 4 to about 5, about 5 to about 7, about 5 toabout 6, or about 6 to about 7. In some embodiments, n is 1, 2, 3 or 4.

In one illustrative example, a hydroxypropyl chain of the structure—CH₂CH(CH₃)OH includes one hydroxypropyl group in the hydroxypropylchain (i.e., n=1). In another illustrative example a hydroxypropyl chainof the structure —[CH₂CH(CH₃)O]₃H includes three hydroxypropyl groups inthe hydroxypropyl chain (i.e., n=3).

In certain embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 3.3±0.3, 3.4±0.3, 3.6±0.3, or 3.8±0.3. In otherembodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 4.0±0.3, 4.2±0.3, 4.4±0.3, 4.6±0.3, or 4.8±0.3. Inother embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 5.0±0.3, 5.2±0.3, 5.4±0.3, 5.6±0.3, or 5.8±0.3. Andin other embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 6.0±0.3, 6.2±0.3, 6.4±0.3, 6.6±0.3, or 6.7±0.3.

In some embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 3.2±0.2, 3.3±0.2, 3.4±0.2, 3.5±0.2, 3.6±0.2,3.7±0.2, or 3.8±0.2. In other embodiments, the average number ofhydroxypropyl chains per beta-cyclodextrin is 4.0±0.2, 4.1±0.2, 4.2±0.2,4.3±0.2, 4.4±0.2, 4.5±0.2, 4.6±0.2, 4.7±0.2, or 4.8±0.2. In otherembodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 5.0±0.2, 5.1±0.2, 5.2±0.2, 5.3±0.2, 5.4±0.2,5.5±0.2, 5.6±0.2, 5.7±0.2, or 5.8±0.2. And in other embodiments, theaverage number of hydroxypropyl chains per beta-cyclodextrin is 6.0±0.2,6.1±0.2, 6.2±0.2, 6.3±0.2, 6.4±0.2, 6.5±0.2, 6.6±0.2, 6.7±0.2, or6.8±0.2.

In some embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 3.1±0.1, 3.2±0.1, 3.3±0.1, 3.4±0.1, 3.5±0.1,3.6±0.1, 3.7±0.1, 3.8±0.1, or 3.9±0.1. In other embodiments, the averagenumber of hydroxypropyl chains per beta-cyclodextrin is 4.0±0.1,4.1±0.1, 4.2±0.1, 4.3±0.1, 4.4±0.1, 4.5±0.1, 4.6±0.1, 4.7±0.1, 4.8±0.1,or 4.9±0.1. In other embodiments, the average number of hydroxypropylchains per beta-cyclodextrin is 5.0±0.1, 5.1±0.1, 5.2±0.1, 5.3±0.1,5.4±0.1, 5.5±0.1, 5.6±0.1, 5.7±0.1, 5.8±0.1, or 5.9±0.1. And in otherembodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is 6.0±0.1, 6.1±0.1, 6.2±0.1, 6.3±0.1, 6.4±0.1,6.5±0.1, 6.6±0.1, 6.7±0.1, 6.8±0.1, or 6.9±0.1.

In some embodiments, at least about 50%, e.g., about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, or about 97%, of the hydroxypropyl chains have n=1. In someembodiments, at least 70% of the hydroxypropyl chains have n=1. In someembodiments, at least 90% of the hydroxypropyl chains have n=1.

In some embodiments, percentage of the hydroxypropyl chains that haven=1 ranges from about 50% to about 99%, such as about 55% to about 99%,about 60% to about 99%, about 65% to about 99%, about 70% to about 99%,about 75% to about 99%, about 80% to about 99%, about 85% to about 99%,about 90% to about 99%, about 95% to about 99%; such as from about 50%to about 97%, such as about 55% to about 97%, about 60% to about 97%,about 65% to about 97%, about 70% to about 97%, about 75% to about 97%,about 80% to about 97%, about 85% to about 97%, about 90% to about 97%,about 95% to about 97%; such as from about 50% to about 95%, about 55%to about 95%, about 60% to about 95%, about 65% to about 95%, about 70%to about 95%, about 75% to about 95%, about 80% to about 95%, about 85%to about 95%, about 90% to about 95%; such as from about 50% to about90%, about 55% to about 90%, about 60% to about 90%, about 65% to about90%, about 70% to about 90%, about 75% to about 90%, about 80% to about90%, about 85% to about 90%; such as from about 50% to about 85%, about55% to about 85%, about 60% to about 85%, about 65% to about 85%, about70% to about 85%, about 75% to about 85%, about 80% to about 85%; suchas from about 50% to about 80%, about 55% to about 80%, about 60% toabout 80%, about 65% to about 80%, about 70% to about 80%, about 75% toabout 80%; such as from about 50% to about 75%, about 55% to about 75%,about 60% to about 75%, about 65% to about 75%, about 70% to about 75%;such as from about 50% to about 70%, about 55% to about 70%, about 60%to about 70%, about 65% to about 70%; such as from about 50% to about65%, about 55% to about 65%, about 60% to about 65%; such as from about50% to about 60%, about 55% to about 60%; or such as from about 50% toabout 55%.

In some embodiments, less than about 50%, such as about 45%, about 40%,about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about5%, or about 3%, of the hydroxypropyl chains have n=2. In someembodiments, less than 30% of the hydroxypropyl chains have n=2. In someembodiments, less than 10% of the hydroxypropyl chains have n=2.

In some embodiments, the percentage of the hydroxypropyl chains thathave n=2 ranges from about 5% to about 50%, such as about 10% to about50%, about 15% to about 50%, about 20% to about 50%, about 25% to about50%, about 30% to about 50%, about 35% to about 50%, about 40% to about50%, about 45% to about 50%; such as from about 5% to about 45%, about10% to about 45%, about 15% to about 45%, about 20% to about 45%, about25% to about 45%, about 30% to about 45%, about 35% to about 45%, about40% to about 45%; such as from about 5% to about 40%, about 10% to about40%, about 15% to about 40%, about 20% to about 40%, about 25% to about40%, about 30% to about 40%, about 35% to about 40%; such as from about5% to about 35%, about 10% to about 35%, about 15% to about 35%, about20% to about 35%, about 25% to about 35%, about 30% to about 35%; suchas from about 5% to about 30%, about 10% to about 30%, about 15% toabout 30%, about 20% to about 30%, about 25% to about 30%; such as fromabout 5% to about 25%, about 10% to about 25%, about 15% to about 25%,about 20% to about 25%; such as from about 5% to about 20%, about 10% toabout 20%, about 15% to about 20%; such as from about 5% to about 15%,about 10% to about 15%; or about 5% to about 10%.

In some embodiments, less than about 50%, such as about 45%, about 40%,about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about5%, or about 3%, of the hydroxypropyl chains have n>2. In someembodiments, less than 10% of the hydroxypropyl chains have n>2.

In some embodiments, the percentage of the hydroxypropyl chains thathave n>2 ranges from about 5% to about 50%, such as about 10% to about50%, about 15% to about 50%, about 20% to about 50%, about 25% to about50%, about 30% to about 50%, about 35% to about 50%, about 40% to about50%, about 45% to about 50%; such as from about 5% to about 45%, about10% to about 45%, about 15% to about 45%, about 20% to about 45%, about25% to about 45%, about 30% to about 45%, about 35% to about 45%, about40% to about 45%; such as from about 5% to about 40%, about 10% to about40%, about 15% to about 40%, about 20% to about 40%, about 25% to about40%, about 30% to about 40%, about 35% to about 40%; such as from about5% to about 35%, about 10% to about 35%, about 15% to about 35%, about20% to about 35%, about 25% to about 35%, about 30% to about 35%; suchas from about 5% to about 30%, about 10% to about 30%, about 15% toabout 30%, about 20% to about 30%, about 25% to about 30%; such as fromabout 5% to about 25%, about 10% to about 25%, about 15% to about 25%,about 20% to about 25%; such as from about 5% to about 20%, about 10% toabout 20%, about 15% to about 20%; such as from about 5% to about 15%,about 10% to about 15%; or such as from about 5% to about 10%.

In some embodiments, the average number of hydroxypropyl chains perbeta-cyclodextrin is about 4 to about 6. In some embodiments, at leastabout 60%, such as at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 97%, of the beta-cyclodextrinscollectively have an average number of hydroxypropyl chains perbeta-cyclodextrin of about 4 to about 6. In some embodiments, thepercentage of the beta-cyclodextrins that collectively have an averagenumber of hydroxypropyl chains per beta-cyclodextrin of about 4 to about6 ranges from about 60% to about 97%, such as about 65% to about 97%,about 70% to about 97%, about 75% to about 97%, about 80% to about 97%,about 85% to about 97%, about 90% to about 97%; such as from about 60%to about 95%, about 65% to about 95%, about 70% to about 95%, about 75%to about 95%, about 80% to about 95%, about 85% to about 95%, about 90%to about 95%; such as from about 60% to about 90%, about 65% to about90%, about 70% to about 90%, about 75% to about 90%, about 80% to about90%, about 85% to about 90%; such as from about 60% to about 85%, about65% to about 85%, about 70% to about 85%, about 75% to about 85%, about80% to about 85%; such as from about 60% to about 80%, about 65% toabout 80%, about 70% to about 80%, about 75% to about 80%; such as fromabout 60% to about 75%, about 65% to about 75%, about 70% to about 75%;such as from about 60% to about 70%, about 65% to about 70%; or such asfrom about 60% to about 65%.

6.2.1.2. Substitution Fingerprint

6.2.1.2.1. DS-0

In typical embodiments, the pharmaceutically active ingredient containsless than about 2%, such as less than about 1.5%, less than about 1.4%,less than about 1.3%, less than about 1.2%, less than about 1.1%, lessthan about 1.0%, less than about 0.9%, less than about 0.8%, less thanabout 0.7%, less than about 0.6%, less than about 0.5%, less than about0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%,less than about 0.09%, less than about 0.08%, less than about 0.07%,less than about 0.06%, or less than about 0.05% unsubstitutedbeta-cyclodextrin (“DS-0”; “BCD”), as determined by peak height of anelectrospray MS spectrum.

In typical embodiments, no more than (“NMT”) 1% of the beta-cyclodextrinmixture is unsubstituted with a hydroxypropyl group (BCD), as determinedby peak height of an electrospray MS spectrum.

In some embodiments, the level of unsubstituted beta-cyclodextrins inthe pharmaceutically active ingredient ranges from about 0.05% to about2%, such as about 0.05% to about 1.5%, about 0.05% to about 1.4%, about0.05% to about 1.3%, about 0.05% to about 1.2%, about 0.05% to about1.1%, about 0.05% to about 1.0%, about 0.05% to about 0.8%, about 0.05%to about 0.6%, about 0.05% to about 0.5%, about 0.05% to about 0.4%,about 0.05% to about 0.3%, about 0.05% to about 0.2%, about 0.05% toabout 0.1%, about 0.05% to about 0.07%, about 0.07% to about 1.5%, about0.07% to about 1.4%, about 0.07% to about 1.3%, about 0.07% to about1.2%, about 0.07% to about 1.1%, about 0.07% to about 1.0%, about 0.07%to about 0.8%, about 0.07% to about 0.6%, about 0.07% to about 0.5%,about 0.07% to about 0.4%, about 0.07% to about 0.3%, about 0.07% toabout 0.2%, about 0.07% to about 0.1%, about 0.1% to about 1.5%, about0.1% to about 1.4%, about 0.1% to about 1.3%, about 0.1% to about 1.2%,about 0.1% to about 1.1%, about 0.1% to about 1.0%, about 0.1% to about0.8%, about 0.1% to about 0.6%, about 0.1% to about 0.5%, about 0.1% toabout 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about0.2% to about 1.5%, about 0.2% to about 1.4%, about 0.2% to about 1.3%,about 0.2% to about 1.2%, about 0.2% to about 1.1%, about 0.2% to about1.0%, about 0.2% to about 0.8%, about 0.2% to about 0.6%, about 0.2% toabout 0.5%, about 0.2% to about 0.4%, about 0.2% to about 0.3%, about0.3% to about 1.5%, about 0.3% to about 1.4%, about 0.3% to about 1.3%,about 0.3% to about 1.2%, about 0.3% to about 1.1%, about 0.3% to about1.0%, about 0.3% to about 0.8%, about 0.3% to about 0.6%, about 0.3% toabout 0.5%, about 0.3% to about 0.4%, about 0.4% to about 1.5%, about0.4% to about 1.4%, about 0.4% to about 1.3%, about 0.4% to about 1.2%,about 0.4% to about 1.1%, about 0.4% to about 1.0%, about 0.4% to about0.8%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, about 0.5% toabout 1.5%, about 0.5% to about 1.4%, about 0.5% to about 1.3%, about0.5% to about 1.2%, about 0.5% to about 1.1%, about 0.5% to about 1.0%,about 0.5% to about 0.8%, about 0.5% to about 0.6%, about 0.6% to about1.5%, about 0.6% to about 1.4%, about 0.6% to about 1.3%, about 0.6% toabout 1.2%, about 0.6% to about 1.1%, about 0.6% to about 1.0%, about0.6% to about 0.8%, about 0.8% to about 1.5%, about 0.8% to about 1.4%,about 0.8% to about 1.3%, about 0.8% to about 1.2%, about 0.8% to about1.1%, about 0.8% to about 1.0%, about 1.0% to about 1.5%, about 1.0% toabout 1.4%, about 1.0% to about 1.3%, about 1.0% to about 1.2%, about1.0% to about 1.1%, about 1.1% to about 1.5%, about 1.1% to about 1.4%,about 1.1% to about 1.3%, about 1.1% to about 1.2%, about 1.2% to about1.5%, about 1.2% to about 1.4%, about 1.2% to about 1.3%, about 1.3% toabout 1.5%, about 1.3% to about 1.4%, or about 1.4% to about 1.5%.

In various embodiments, the composition comprises no more than about0.01% BCD, no more than about 0.02% BCD, no more than about 0.03% BCD,no more than about 0.04% BCD, or no more than about 0.05% BCD of thebeta-cyclodextrin mixture.

6.2.1.2.2. DS-1

In typical embodiments, less than 4% of the beta-cyclodextrin mixture isbeta-cyclodextrin substituted with just one hydroxypropyl group(“DS-1”), as determined by peak height of an electrospray MS spectrum.

In various embodiments, less than 3.9%, less than 3.8%, less than 3.7%,less than 3.6%, or less than 3.5% of the beta-cyclodextrin mixture isDS-1. In certain embodiments, the pharmaceutically active ingredientcomprises less than 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, or 3.0% DS-1. Inparticular embodiments, the pharmaceutically active ingredient comprisesless than 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, or 2.0%DS-1. In some embodiments, the mixture of beta-cyclodextrin moleculescomprises less than 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%,1.1%, or 1.0% DS-1. In presently preferred embodiments, thepharmaceutically active ingredient comprises less than 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% DS-1, even less than about 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03% DS-1. In certain preferredembodiments, the composition comprises less than 0.03%, even as low as0.02% DS-1.

In currently preferred embodiments, the composition comprises less thanabout 0.05% BCD and less than about 0.03% DS-1.

6.2.1.2.3. DS-2

In various embodiments, the beta-cyclodextrin mixture has a lowpercentage of beta-cyclodextrin substituted with two hydroxypropylgroups (“DS-2”), as determined by peak height of an electrospray MSspectrum.

In various embodiments, less than 3.9%, less than 3.8%, less than 3.7%,less than 3.6%, or less than 3.5% of the beta-cyclodextrin mixture isDS-2. In certain embodiments, the pharmaceutically active ingredientcomprises less than 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, or 3.0% DS-2. Inparticular embodiments, the pharmaceutically active ingredient comprisesless than 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, or 2.0%DS-2. In some embodiments, the mixture of beta-cyclodextrin moleculescomprises less than 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%,1.1%, or 1.0% DS-2. In presently preferred embodiments, thepharmaceutically active ingredient comprises less than 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% DS-2, even less than about 0.09%,0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03% DS-2. In certain preferredembodiments, the composition comprises less than 0.03%, even as low as0.02% DS-2.

6.2.1.2.4. DS-3

In typical embodiments, the mixture comprises at least 10%beta-cyclodextrin molecules having three hydroxypropyl substitutions(“DS-3”) as a percentage of the total mixture, as determined by peakheight of an electrospray MS spectrum. In various embodiments, at least11%, at least 12%, at least 13%, at least 14%, at least 15%, at least16%, at least 17%, at least 18%, at least 19%, at least 20%, at least21%, at least 22%, at least 23%, at least 24%, or at least 25% of thebeta-cyclodextrin mixture is DS-3.

In various embodiments, the mixture comprises no more than 30%, such asno more than 29%, no more than 28%, no more than 27%, no more than 26%,no more than 25%, no more than 24%, no more than 23%, no more than 22%,no more than 21%, no more than 20%, no more than 19%, no more than 18%,no more than 17%, no more than 16%, or no more than 15% of DS-3 as apercentage of the total mixture.

In certain embodiments, the percentage of DS-3 in the entire mixtureranges from 10% to 30%, such as 11% to 29%, 12% to 28%, 13% to 27%, 14%to 26%, 15% to 25%, 16% to 24%, 17% to 23%, 18% to 22%, or 19% to 21%.

In various embodiments, the mixture comprises about 15%, about 16%,about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about23%, about 24%, or about 25% DS-3.

6.2.1.2.5. DS-4

In typical embodiments, the mixture comprises at least 20%beta-cyclodextrin molecules having four hydroxypropyl substitutions(“DS-4”) as a percentage of the total mixture, as determined by peakheight of an electrospray MS spectrum. In various embodiments, at least21%, at least 22%, at least 23%, at least 24%, at least 25%, at least26%, at least 27%, at least 28%, at least 29%, at least 30%, at least31%, at least 32%, at least 33%, at least 34%, or at least 35% of thebeta-cyclodextrin mixture is DS-4.

In various embodiments, the mixture comprises no more than 40%, such asno more than 39%, no more than 38%, no more than 37%, no more than 36%,no more than 35%, no more than 34%, no more than 33%, no more than 32%,no more than 31%, no more than 30%, no more than 29%, no more than 28%,no more than 27%, no more than 26%, or no more than 25% of DS-4 as apercentage of the total mixture.

In certain embodiments, the percentage of DS-4 in the entire mixtureranges from 20% to 40%, such as 21% to 39%, 22% to 38%, 23% to 37%, 24%to 36%, 25% to 35%, 26% to 34%, 27% to 33%, 28% to 32%, or 29% to 31%.

In various embodiments, the mixture comprises about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, or about 35% DS-4.

6.2.1.2.6. DS-5

In typical embodiments, the mixture comprises at least 15%beta-cyclodextrin molecules having five hydroxypropyl substitutions(“DS-5”) as a percentage of the total mixture, as determined by peakheight of an electrospray MS spectrum. In various embodiments, at least16%, at least 17%, at least 18%, at least 19%, at least 20%, at least21%, at least 22%, at least 23%, at least 24%, at least 25%, at least26%, at least 27%, at least 28%, at least 29%, or at least 30% of thebeta-cyclodextrin mixture is DS-5.

In various embodiments, the mixture comprises no more than 35%, such asno more than 34%, no more than 33%, no more than 32%, no more than 31%,no more than 30%, no more than 29%, no more than 28%, no more than 27%,no more than 26%, no more than 25%, no more than 24%, no more than 23%,no more than 22%, no more than 21%, or no more than 20% of DS-5 as apercentage of the total mixture.

In certain embodiments, the percentage of DS-5 in the entire mixtureranges from 15% to 35%, such as 16% to 34%, 17% to 33%, 18% to 32%, 19%to 31%, 20% to 30%, 21% to 29%, 22% to 28%, 23% to 27%, or 24% to 26%.

In various embodiments, the mixture comprises about 20%, about 21%,about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about28%, about 29%, or about 30% DS-5.

6.2.1.2.7. DS-6

In typical embodiments, the mixture comprises at least 5%beta-cyclodextrin molecules having six hydroxypropyl substitutions(“DS-6”) as a percentage of the total mixture, as determined by peakheight of an electrospray MS spectrum. In various embodiments, at least6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%,at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, atleast 17%, at least 18%, at least 19%, or at least 20% of thebeta-cyclodextrin mixture is DS-6.

In various embodiments, the mixture comprises no more than 25%, such asno more than 24%, no more than 23%, no more than 22%, no more than 21%,no more than 20%, no more than 19%, no more than 18%, no more than 17%,no more than 16%, no more than 15%, no more than 14%, no more than 13%,no more than 12%, no more than 11%, or no more than 10% of DS-6 as apercentage of the total mixture.

In certain embodiments, the percentage of DS-6 in the entire mixtureranges from 5% to 25%, such as 6% to 24%, 7% to 23%, 8% to 22%, 9% to21%, 10% to 20%, 11% to 19%, 12% to 18%, 13% to 17%, or 14% to 16%.

In various embodiments, the mixture comprises about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,about 16%, or about 17% DS-6.

6.2.1.2.8. DS-7

In typical embodiments, less than 10% of the beta-cyclodextrin mixtureis beta-cyclodextrin substituted with seven hydroxypropyl groups(“DS-7”) as a percentage of the total mixture, as determined by peakheight of an electrospray MS spectrum. In various embodiments, less than9%, less than 8%, less than 7%, less than 6%, less than 5%, less than4%, less than 3%, less than 2%, or less than 1% of the beta-cyclodextrinmixture is DS-7.

In certain embodiments, the percentage of DS-7 in the entire mixtureranges from 1% to 10%, such as 2% to 9%, 3% to 8%, 4% to 7%, or 5% to6%.

In various embodiments, the mixture comprises about 10%, about 9%, about8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about1% DS-7.

6.2.1.2.9. DS-8

In typical embodiments, the beta-cyclodextrin mixture comprises lessthan 2% of beta-cyclodextrin substituted with eight hydroxypropyl groups(“DS-8”) as a percentage of the total mixture, as determined by peakheight of an electrospray MS spectrum. In various embodiments, less than1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%,less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%,less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, lessthan 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than0.02%, or less than 0.01% of beta-cyclodextrin is DS-8.

In certain embodiments, the percentage of DS-8 in the entire mixtureranges from 0.01% to 2%, such as 0.02% to 1.9%, 0.03% to 1.8%, 0.04% to1.7%, 0.05% to 1.6%, 0.06% to 1.5%, 0.07% to 1.4%, 0.08% to 1.3%, 0.09%to 1.2%, 0.1% to 1.1%, 0.2% to 1%, 0.3% to 0.9%, 0.4% to 0.8%, or 0.5%to 0.7%.

In various embodiments, the mixture comprises about 1.5%, about 1.4%,about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%,about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%,about 0.1%, about 0.09%, about 0.08%, about 0.07%, about 0.06%, about0.05%, about 0.04%, about 0.03%, about 0.02%, or about 0.01% DS-8.

6.2.1.2.10. DS-9

In typical embodiments, no more than 1% of the beta-cyclodextrin mixtureis beta-cyclodextrin substituted with nine hydroxypropyl groups (“DS-9”)as a percentage of the total mixture, as determined by peak height of anelectrospray MS spectrum. In various embodiments, no more than 0.9%, nomore than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%,no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than0.1%, no more than 0.09%, no more than 0.08%, no more than 0.07%, nomore than 0.06%, no more than 0.05%, no more than 0.04%, no more than0.03%, no more than 0.02%, or no more than 0.01% of beta-cyclodextrin isDS-9.

6.2.1.2.11. DS-10

In typical embodiments, no more than 1% of the beta-cyclodextrin mixtureis beta-cyclodextrin substituted with ten hydroxypropyl groups (“DS-10”)as a percentage of the total mixture, as determined by peak height of anelectrospray MS spectrum. In various embodiments, no more than 0.9%, nomore than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%,no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than0.1%, no more than 0.09%, no more than 0.08%, no more than 0.07%, nomore than 0.06%, no more than 0.05%, no more than 0.04%, no more than0.03%, no more than 0.02%, or no more than 0.01% of beta-cyclodextrin isDS-10.

6.2.1.2.12. Profile

In various embodiments, the beta-cyclodextrin mixture contains at least75% of DS-3, DS-4, DS-5, and DS-6, collectively, as a percentage of theentire mixture. In certain embodiments, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, or atleast 98% of beta-cyclodextrin in the mixture is DS-3, DS-4, DS-5, andDS-6, collectively.

In various embodiments, the beta-cyclodextrin mixture comprises DS-3,DS-4, DS-5, and DS-6, collectively, as a percentage of the entiremixture in the range from about 75% to about 98%, such as about 76% toabout 97%, about 77% to about 96%, about 78% to about 95%, about 79% toabout 94%, about 80% to about 93%, about 81% to about 92%, about 82% toabout 91%, about 83% to about 90%, about 84% to about 89%, about 85% toabout 88%, or about 86% to about 87%.

In typical embodiments, the beta-cyclodextrin mixture comprises at least25% of DS-5 and DS-6, collectively, as a percentage of the entiremixture. In certain embodiments, at least 26%, at least 27%, at least28%, at least 29%, at least 30%, at least 31%, at least 32%, at least33%, at least 34%, at least 35%, at least 36%, at least 37%, at least38%, at least 39%, at least 40%, at least 41%, at least 42%, at least43%, at least 44%, at least 45%, at least 46%, at least 47%, at least48%, at least 49%, or at least 50% of beta-cyclodextrin in the mixtureis DS-5 and DS-6, collectively.

In various embodiments, the beta-cyclodextrin mixture comprises DS-5 andDS-6, collectively, as a percentage of the entire mixture in the rangefrom about 25% to about 50%, such as about 26% to about 49%, about 27%to about 48%, about 28% to about 47%, about 29% to about 46%, about 30%to about 45%, about 31% to about 44%, about 32% to about 43%, about 33%to about 42%, about 34% to about 41%, about 35% to about 40%, about 36%to about 39%, or about 37% to about 38%.

In various embodiments, the beta-cyclodextrin species with the greatestprevalence as a percentage of the entire mixture is DS-4.

6.2.1.3. Starting material

In certain embodiments, the present disclosure describes apharmaceutical composition wherein the sole pharmaceutically activeingredient is obtained by purifying one or more hydroxypropylbeta-cyclodextrin products selected from Kleptose® HBP, Kleptose® HP,Trappsol® Cyclo, and Cavasol® W7 HP Pharma.

Kleptose® HBP and Kleptose® HP are hydroxypropyl beta-cyclodextrinproducts available from Roquette Pharma, Lestrem, France. Kleptose® HBPis a parenteral grade endotoxin-controlled composition of hydroxypropylbeta-cyclodextrins with a DS_(a) of about 4.3. Kleptose® HP is anendotoxin-controlled composition of hydroxypropyl beta-cyclodextrinswith a higher DS_(a) than Kleptose® HBP. Trappsol® Cyclo is a parenteralgrade of hydroxypropyl beta cyclodextrin with a DS_(a) of about 6.37,and is available in a powdered or sterile liquid form from SphingoBiotechnology, Inc., a division of CTD Holdings, Inc., Alachua, Fla.,USA. Cavasol® W7 HP Pharma is a pharmaceutical gradehydroxypropyl-beta-cyclodextrin with a DS_(a) from about 4.1 to about5.1, e.g., a DS_(a) of about 4.5, available from Wacker Chemie AG,Munchen, Germany.

In an illustrative example, the pharmaceutical composition is onewherein the sole pharmaceutically active ingredient is obtained bypurifying Kleptose® HBP. In certain embodiments, the pharmaceuticalcomposition is one in which the sole pharmaceutically active ingredientis obtained by purifying Kleptose® HBP (Roquette) by hydrophilicinteraction, e.g., by HPLC purification, or by affinity purification,e.g., affinity chromatography. In various embodiments, thepharmaceutically active ingredient is obtained by purifying Kleptose®HBP (Roquette) by one or more of the procedures described in Examples 6,7 and 9 herein.

In a variety of embodiments, the purification provides a portion orfraction of the Kleptose® HBP having increased activity, e.g., increasedaffinity for unesterified cholesterol.

In other embodiments, the pharmaceutical composition is one in which thesole pharmaceutically active ingredient is obtained by purifyingTrappsol® Cyclo (CTD) by hydrophilic interaction, e.g., by HPLCpurification, or by affinity purification, e.g., affinitychromatography. In various embodiments, the pharmaceutically activeingredient is obtained by purifying Trappsol® Cyclo with absorptionchromatography on alumina using one or more of the procedures describedin Examples 6, 7 and 9 herein.

In some embodiments, the pharmaceutical composition purified fromTrappsol® Cyclo (CTD) comprises a mixture of beta-cyclodextrin moleculessubstituted at one or more hydroxyl positions by hydroxypropyl groups,the mixture optionally including unsubstituted beta-cyclodextrinmolecules, and a diluent that is pharmaceutically acceptable forintrathecal, intracerebroventricular, or intravenous administration. Thecomposition comprises no more than (“NMT”) 5 EU of endotoxins per gramof beta-cyclodextrin mixture, no more than 0.5% propylene glycol, asmeasured by the HPLC method set forth in the USP Hydroxypropyl Betadexmonograph, and no more than 1 ppm propylene oxide, determined accordingto the USP Hydroxypropyl Betadex monograph.

In some embodiments, the pharmaceutical composition purified fromTrappsol® Cyclo (CTD) comprises NMT 1.5 EU of endotoxins per gram ofbeta-cyclodextrin mixture. In some embodiments, the compositioncomprises no more than 0.01% propylene glycol, as measured by the HPLCmethod set forth in the USP Hydroxypropyl Betadex monograph.

In certain embodiments, the pharmaceutically active ingredient purifiedfrom Trappsol® Cyclo (CTD) comprises less than 5%, such as less than4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, lessthan 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1%unsubstituted beta-cyclodextrin (“DS-0”), beta-cyclodextrin substitutedwith one hydroxypropyl group (“DS-1”), and beta-cyclodextrin substitutedwith two hydroxypropyl groups (“DS-2”), collectively, as determined bypeak heights of an electrospray MS spectrum.

In certain embodiments, the pharmaceutically active ingredient purifiedfrom Trappsol® Cyclo (CTD) comprises at least 50%, such as at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, or at least 90% beta-cyclodextrin substituted with fivehydroxypropyl groups (“DS-5”), beta-cyclodextrin substituted with sixhydroxypropyl groups (“DS-6”), and beta-cyclodextrin substituted withseven hydroxypropyl groups (“DS-7”), collectively, as determined by peakheights of an electrospray MS spectrum.

In certain embodiments, the pharmaceutically active ingredient purifiedfrom Trappsol® Cyclo (CTD) comprises less than 5%, such as less than4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, lessthan 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1%beta-cyclodextrin substituted with nine hydroxypropyl groups (“DS-9”)and beta-cyclodextrin substituted with ten hydroxypropyl groups(“DS-10”), collectively, as determined by peak heights of anelectrospray MS spectrum.

In still other embodiments, the pharmaceutical composition is one inwhich the sole pharmaceutically active ingredient is obtained bypurifying Cavasol® W7 HP by hydrophilic interaction, e.g., by HPLCpurification, or by affinity purification, e.g., affinitychromatography. In various embodiments, the pharmaceutically activeingredient is obtained by purifying Cavasol® W7 HP by one or more of theprocedures described in Examples 6, 7 and 9 herein.

In certain embodiments, purifying one or more hydroxypropylbeta-cyclodextrin products selected from Kleptose® HBP, Kleptose® HP,Trappsol® Cyclo, and Cavasol® W7 HP Pharma comprises one or more ofcomplex formation, precipitation, and adsorption chromatography. In someembodiments, the purification comprises one method, e.g., adsorptionchromatography. In some embodiments, the purification comprises two ormore methods, e.g., precipitation in combination with adsorptionchromatography. In cases where the purification comprises two or moremethods used in combination, the methods can be combined in any order topurify a hydroxypropyl beta-cyclodextrin product. In an illustrativeexample, Kleptose® HBP or Trappsol® Cyclo can first be subjected toadsorption chromatography, then one or more selected fractions from thechromatographic step can be subjected to solvent precipitation from aprecipitation solvent system to effect further purification. In analternative example, Kleptose® HBP can first be subjected to solventprecipitation from a precipitation solvent system, then the precipitatecan be subjected to adsorption chromatography to effect furtherpurification.

In some embodiments, the purification of one or more hydroxypropylbeta-cyclodextrin products, e.g., Kleptose® HBP or Trappsol® Cyclo,results in an increase in DS_(a) due to removal of unsubstituted (DS=0)and/or monosubstituted (DS=1) beta-cyclodextrins. In an illustrativeexample, a commercial sample of Kleptose® HBP having DS_(a)=4.34contains 0.6% unsubstituted beta-cyclodextrins (DS=0) and 3.68%monosubstituted beta-cyclodextrins (DS=1). The DS_(a) after removal ofthe DS=0 and DS=1 species can be calculated using the followingequations:

x(0)+y(1)+z(DS _(a))=4.34

x+y+z=1

wherein x=fraction of unsubstituted beta-cyclodextrins; y=fraction ofmonosubstituted beta-cyclodextrins; z=fraction of beta-cyclodextrinswith DS≥2. In this instance, the DS_(a) of the resulting sample afterremoval of beta-cyclodextrins having DS=0 and DS=1 is 4.5.

Accordingly, in certain embodiments the present disclosure provides amethod for purifying one or more hydroxypropyl beta-cyclodextrinproducts selected from Kleptose® HBP, Kleptose® HP, Trappsol® Cyclo, andCavasol® W7 HP Pharma, particularly Kleptose® HBP or Trappsol® Cyclo,wherein the purification method reduces in the product the amount ofpropylene glycol or propylene glycol oligomers (e.g., by solventprecipitation) and/or the amount of unsubstituted beta-cyclodextrin(DS=0) and/or the amount of monosubstituted beta-cyclodextrin (DS=1)(e.g., by adsorption chromatography). In certain of such embodiments,wherein the amount of unsubstituted beta-cyclodextrin (DS=0) and/or theamount of monosubstituted beta-cyclodextrin (DS=1) in the product isreduced, the purified product exhibits an increased DS_(a). Hence, incertain embodiments the present disclosure provides a method forincreasing the DS_(a) of one or more hydroxypropyl beta-cyclodextrinproducts selected from Kleptose® HBP, Kleptose® HP, Trappsol® Cyclo, andCavasol® W7 HP Pharma, particularly Kleptose® HBP or Trappsol® Cyclo,the method comprising reducing the amount of unsubstitutedbeta-cyclodextrin (DS=0) and/or monosubstituted beta-cyclodextrin (DS=1)in the product, for example, according one or more purification stepsdescribed herein, such as adsorption chromatography.

Although Kleptose® HBP, Kleptose® HP, Trappsol® Cyclo, and Cavasol® W7HP Pharma each have a reported DS_(a), as discussed above, DS_(a) is anaverage measure, and therefore each of these hydroxypropylbeta-cyclodextrin products is comprised of hydroxypropylbeta-cyclodextrins with varying DS values. In some embodiments, thepharmaceutically active ingredient described herein is obtained byisolating from one or more of these products one or more hydroxypropylbeta-cyclodextrin fractions with a DS_(a) described herein.

6.2.2. Endotoxin Levels

In certain embodiments, the pharmaceutical compositions of thedisclosure contain a low level of bacterial endotoxins. The low level ofbacterial endotoxins allows for the disclosed pharmaceuticalcompositions to be administered by certain routes, e.g., via intrathecalor intracerebroventricular administration, for longer periods and athigher levels than can safely be done with other compositions havinghigher levels of endotoxins.

As used herein, “IU” refers to an International Unit of bacterialendotoxin, also known as a United States Pharmacopeial (USP) EndotoxinUnit (“EU”). The level of bacterial endotoxins (IU; synonymously, EU) inthe composition is determined by Limulus amoebocyte lysate test,according to the procedures set forth in “<85> Bacterial EndotoxinsTest,” the United States Pharmacopeial Convention, Interim RevisionAnnouncement dated Apr. 1, 2011 (“USP Endotoxin Monograph”),incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutical composition contains less thanabout 10 IU, such as less than about 6, about 5, about 4, about 3, about2, about 1.5, about 1.2, about 1 IU, about 0.8 IU, about 0.6 IU, about0.5 IU, about 0.4 IU, about 0.3 IU, about 0.2 IU, about 0.1 IU, about0.07 IU, or about 0.05 IU endotoxin per gram of pharmaceutically activeingredient. In some embodiments, the pharmaceutical composition containsa level of bacterial endotoxin in a range of from about 0.05 IU to about10 IU, e.g., about 0.05 IU to about 6 IU, about 0.05 IU to about 5 IU,about 0.05 IU to about 4 IU, about 0.05 IU to about 3 IU, about 0.05 IUto about 2 IU, about 0.05 IU to about 1.5 IU, about 0.05 IU to about 1.2IU, about 0.05 IU to about 1 IU, about 0.05 IU to about 0.8 IU, about0.05 IU to about 0.6 IU, about 0.05 IU to about 0.5 IU, about 0.05 IU toabout 0.4 IU, about 0.05 IU to about 0.3 IU, about 0.05 IU to about 0.2IU, or about 0.05 IU to about 0.1 IU endotoxin per gram of thebeta-cyclodextrin mixture.

In certain embodiments, the pharmaceutical composition comprises no morethan (“NMT”) 5 EU/g beta-cyclodextrin mixture, NMT 4 EU/gbeta-cyclodextrin mixture, NMT 3 EU/g beta-cyclodextrin mixture, or nomore than 2 EU/g beta-cyclodextrin mixture. In preferred embodiments,the pharmaceutical composition comprises NMT 1.5 EU/g beta-cyclodextrinmixture. In certain embodiments, the pharmaceutical compositioncomprises NMT 1.4 EU/g beta-cyclodextrin mixture, NMT 1.3 EU/gbeta-cyclodextrin mixture, NMT 1.2 EU/g beta-cyclodextrin mixture, NMT1.1 EU/g beta-cyclodextrin mixture, or NMT 1.0 EU/g beta-cyclodextrinmixture.

6.2.3. Process Impurities

Pharmaceutical compositions comprising mixtures of hydroxypropylbeta-cyclodextrins may contain impurities arising from the synthesis ofhydroxypropyl beta-cyclodextrins. Such impurities may include unreactedstarting materials such as unsubstituted beta-cyclodextrins andpropylene oxide, and reaction by-products such as propylene glycol andpropylene glycol oligomers. In certain embodiments, the pharmaceuticalcompositions described herein exhibit reduced levels of one or more ofsuch impurities.

6.2.3.1. Propylene Glycol

In some embodiments, the pharmaceutically active ingredient comprisesless than about 1%, such as less than about 0.9%, 0.8%, 0.7%, 0.6%, or0.5%, propylene glycol, determined according to the USP HydroxypropylBetadex monograph. In various embodiments, the pharmaceutically activeingredient comprises less than about 0.4%, 0.3%, 0.2% or 0.1% propyleneglycol, determined according to the USP Hydroxypropyl Betadex monograph.In certain embodiments, the pharmaceutical composition comprises lessthan about 0.09%, 0.08%, 0.07%, or less than about 0.05% propyleneglycol, determined according to the USP Hydroxypropyl Betadex monograph.In currently preferred embodiments, the pharmaceutically activeingredient comprises no more than 0.5% propylene glycol, determinedaccording to the USP Hydroxypropyl Betadex monograph.

In some embodiments, the pharmaceutically active ingredient comprisesfrom about 0.05% to about 1% propylene glycol, such as about 0.05% toabout 0.8%, about 0.05% to about 0.6%, about 0.05% to about 0.5%, about0.05% to about 0.4%, about 0.05% to about 0.3%, about 0.05% to about0.2%, about 0.05% to about 0.1%, about 0.05% to about 0.07%, about 0.07%to about 1%, about 0.07% to about 0.8%, about 0.07% to about 0.6%, about0.07% to about 0.5%, about 0.07% to about 0.4%, about 0.07% to about0.3%, about 0.07% to about 0.2%, about 0.07% to about 0.1%, about 0.1%to about 1%, about 0.1% to about 0.8%, about 0.1% to about 0.6%, about0.1% to about 0.5%, about 0.1% to about 0.4%, about 0.1% to about 0.3%,about 0.1% to about 0.2%, about 0.2% to about 1%, about 0.2% to about0.8%, about 0.2% to about 0.6%, about 0.2% to about 0.5%, about 0.2% toabout 0.4%, about 0.2% to about 0.3%, about 0.3% to about 1%, about 0.3%to about 0.8%, about 0.3% to about 0.6%, about 0.3% to about 0.5%, about0.3% to about 0.4%, about 0.4% to about 1%, about 0.4% to about 0.8%,about 0.4% to about 0.6%, about 0.4% to about 0.5%, about 0.5% to about1%, about 0.5% to about 0.8%, about 0.5% to about 0.6%, about 0.6% toabout 1%, about 0.6% to about 0.8%, or about 0.8% to about 1.0%,determined according to the USP Hydroxypropyl Betadex monograph.

In some embodiments, the pharmaceutically active ingredient comprisesless than about 0.01% propylene glycol monomers, determined according tothe USP Hydroxypropyl Betadex monograph. In some embodiments, thepharmaceutically active ingredient comprises less than about 0.2%propylene glycol dimers, determined according to the USP HydroxypropylBetadex monograph. In some embodiments, the pharmaceutically activeingredient comprises less than about 0.2% propylene glycol trimers,determined according to the USP Hydroxypropyl Betadex monograph.

6.2.3.2. Propylene Oxide

In some embodiments, the pharmaceutically active ingredient containsless than about 1 ppm, such as less than about 0.8 ppm, less than about0.6 ppm, less than about 0.5 ppm, less than about 0.4 ppm, less thanabout 0.3 ppm, less than about 0.2 ppm, less than about 0.1 ppm, lessthan about 0.07 ppm, or less than about 0.05 ppm, propylene oxide,determined according to the USP Hydroxypropyl Betadex monograph. Forexample, the pharmaceutically active ingredient can have about 1, about0.8, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1,about 0.07, or about 0.05 ppm propylene oxide, determined according tothe USP Hydroxypropyl Betadex monograph.

In some embodiments, the pharmaceutically active ingredient has anamount of propylene oxide in a range of from about 0.05 to about 1 ppm,such as about 0.05 to about 0.8, about 0.05 to about 0.6, about 0.05 toabout 0.5, about 0.05 to about 0.4, about 0.05 to about 0.3, about 0.05to about 0.2, about 0.05 to about 0.1, about 0.1 to about 1, about 0.1to about 0.8, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1to about 0.4, about 0.1 to about 0.3, about 0.1 to about 0.2, about 0.2to about 1, about 0.2 to about 0.8, about 0.2 to about 0.6, about 0.2 toabout 0.5, about 0.2 to about 0.4, about 0.2 to about 0.3, about 0.3 toabout 1, about 0.3 to about 0.8, about 0.3 to about 0.6, about 0.3 toabout 0.5, about 0.3 to about 0.4, about 0.4 to about 1, about 0.4 toabout 0.8, about 0.4 to about 0.6, about 0.4 to about 0.5, about 0.5 toabout 1, about 0.5 to about 0.8, about 0.5 to about 0.6, about 0.6 toabout 1, about 0.6 to about 0.8, or about 0.8 to about 1 ppm, determinedaccording to the USP Hydroxypropyl Betadex monograph.

6.2.4. Other Compositional Characteristics

Hydroxypropyl beta-cyclodextrin compositions comprising apharmaceutically active ingredient of the disclosure and, optionally,one or more additional therapeutic agents, such as the combinationtherapeutic agents described in Section 6.3.3., are provided herein.

In certain embodiments, the pharmaceutical composition comprises about100 mg to about 2000 mg, such as about 100 to about 1800, about 100 toabout 1600, about 100 to about 1500, about 100 to about 1200, about 100to about 1000, about 100 to about 800, about 100 to about 600, about 100to about 500, about 100 to about 400, about 100 to about 300, about 100to about 200, about 200 to about 2000, about 200 to about 1800, about200 to about 1600, about 200 to about 1500, about 200 to about 1200,about 200 to about 1000, about 200 to about 800, about 200 to about 600,about 200 to about 500, about 200 to about 400, about 200 to about 300,about 300 to about 2000, about 300 to about 1800, about 300 to about1600, about 300 to about 1500, about 300 to about 1200, about 300 toabout 1000, about 300 to about 800, about 300 to about 600, about 300 toabout 500, about 300 to about 400, about 400 to about 2000, about 400 toabout 1800, about 400 to about 1600, about 400 to about 1500, about 400to about 1200, about 400 to about 1000, about 400 to about 800, about400 to about 600, about 400 to about 500, about 500 to about 2000, about500 to about 1800, about 500 to about 1600, about 500 to about 1500,about 500 to about 1200, about 500 to about 1000, about 500 to about800, about 500 to about 600, about 600 to about 2000, about 600 to about1800, about 600 to about 1600, about 600 to about 1500, about 600 toabout 1200, about 600 to about 1000, about 600 to about 800, about 800to about 2000, about 800 to about 1800, about 800 to about 1600, about800 to about 1500, about 800 to about 1200, or about 800 to about 1000mg of the pharmaceutically active ingredient. For example, thepharmaceutical composition can comprise about 100, about 200, about 300,about 400, about 500, about 600, about 800, about 1000, about 1200,about 1400, about 1500, about 1600, about 1800, or about 2000 mg of thepharmaceutically active ingredient.

In some embodiments, the pharmaceutical composition for administration,for example, a pharmaceutical composition suitable for intrathecaladministration, has a concentration of about 10 mg/mL to about 200mg/mL, such as about 10 to about 180, about 10 to about 150, about 10 toabout 120, about 10 to about 100, about 10 to about 80, about 10 toabout 60, about 10 to about 50, about 10 to about 40, about 10 to about30, about 10 to about 20, about 20 to about 200, about 20 to about 180,about 20 to about 150, about 20 to about 120, about 20 to about 100,about 20 to about 80, about 20 to about 60, about 20 to about 50, about20 to about 40, about 20 to about 30, about 30 to about 200, about 30 toabout 180, about 30 to about 150, about 30 to about 120, about 30 toabout 100, about 30 to about 80, about 30 to about 60, about 30 to about50, about 30 to about 40, about 40 to about 200, about 40 to about 180,about 40 to about 150, about 40 to about 120, about 40 to about 100,about 40 to about 80, about 40 to about 60, about 40 to about 50, about50 to about 200, about 50 to about 180, about 50 to about 150, about 50to about 120, about 50 to about 100, about 50 to about 80, about 50 toabout 60, about 60 to about 200, about 60 to about 180, about 60 toabout 150, about 60 to about 120, about 60 to about 100, about 60 toabout 80, about 80 to about 200, about 80 to about 180, about 80 toabout 150, about 80 to about 120, about 80 to about 100, about 100 toabout 200, about 100 to about 180, about 100 to about 150, about 100 toabout 120, about 120 to about 200, about 120 to about 180, about 120 toabout 150, about 150 to about 200, about 150 to about 180, or about 180to about 200 mg/mL of the pharmaceutically active ingredient. Forexample, the pharmaceutical composition can have a concentration ofabout 10, about 20, about 30, about 40, about 50, about 60, about 70,about 80, about 90, about 100, about 110, about 120, about 130, about140, about 150, about 160, about 170, about 180, about 190, or about 200mg/mL of the pharmaceutically active ingredient. In certain embodiments,the pharmaceutical composition for intrathecal administration has aconcentration of about 200 mg/mL of the pharmaceutically activeingredient.

In certain embodiments, the pharmaceutical compositions described hereinexhibit a low level of ototoxicity when administered to an animal. Insome embodiments, the pharmaceutical composition exhibits a lowerototoxicity than Trappsol® Cyclo. The ototoxicity can be assessed invitro by toxicity in a House Ear Institute-organ of Corti 1 (HEI-OC1)cell or in vivo by a brainstem auditory evoked response (BAER) test inan animal, such as a mouse, a rat, a cat, a dog, a monkey, a chimpanzee,or a human See, for example, Leigh-Paffenroth, E. et al. “ObjectiveMeasures of Ototoxicity,” September 2005, vol. 9, No. 1, pages 10-16, inPerspectives on Hearing and Hearing Disorders: Research and Diagnostics,Special Interest Division 6 of the American Speech-Language-HearingAssociation, incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutical composition, e.g., apharmaceutical composition suitable for intrathecal administration, hasan osmolality in a range of from about 300 to about 450 mOsm/kg, e.g.,about 300 to about 400, about 300 to about 350, about 350 to about 450,or about 350 to about 400 mOsm/kg. In some embodiments, the compositionhas an osmolality of about 300, about 320, about 350, about 380, about400, about 420, or about 450 mOsm/kg.

Suitable diluents for pharmaceutical compositions as described herein,e.g., pharmaceutical compositions suitable for intrathecal orintracerebroventricular administration, include isotonic salinesolutions. Compositions, such as pharmaceutical composition suitable forintrathecal administration, may also be diluted with Elliotts B®solution (buffered intrathecal electrolyte/dextrose injection fromLukare Medical, LLC, Scotch Plains, N.J., USA).

In some embodiments, the pharmaceutical composition for injection ismade by dissolving the Active Pharmaceutical Ingredient (the mixture ofbeta-cyclodextrin molecules) in water, adding sodium chloride to 0.9%w/v, and adjusting pH to 6.0-8.0 as necessary with 0.01N sodiumhydroxide. The pharmaceutical composition is then sterile filtered intovials and autoclaved. The product is stable and can be stored at 15-25°C.

The compositions will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. This composition can be in any suitable form(depending upon the desired method of administration). For example, thepharmaceutical composition can be formulated as an aqueous solution andadministered by intrathecal injection or intrathecal infusion.

In some embodiments, pharmaceutical compositions comprise unit doseforms that contain an amount of a pharmaceutically active ingredient ofthe disclosure per dose. Such a unit can contain for example but withoutlimitation about 5 mg to about 5 g, for example 5 mg to about 4 g, 5 mgto about 3 g, 5 mg to about 2 g, 5 mg to about 1 g, about 50 mg to about5 g, about 50 mg to about 4 g, about 50 mg to about 3 g, about 50 mg toabout 2 g, about 50 mg to about 1 g, about 200 mg to about 5 g, about200 mg to about 4 g, about 200 mg to about 3 g, about 200 mg to about 2g, about 200 mg to about 1 g, about 400 mg to about 5 g, about 400 mg toabout 4 g, about 400 mg to about 3 g, about 400 mg to about 2 g, about400 mg to about 1 g, about 500 mg to about 5 g, about 500 mg to about 4g, about 500 mg to about 3 g, about 500 mg to about 2 g, about 500 mg toabout 1 g, about 600 mg to about 5 g, about 600 mg to about 4 g, about600 mg to about 3 g, about 600 mg to about 2 g, about 600 mg to about 1g, about 800 mg to about 5 g, about 800 mg to about 4 g, about 800 mg toabout 3 g, about 800 mg to about 2 g, about 800 mg to about 1 g, about 1g to about 5 g, about 1 g to about 4 g, about 1 g to about 3 g, about 1g to about 2 g, about 1200 mg to about 5 g, about 1200 mg to about 4 g,about 1200 mg to about 3 g, about 1200 mg to about 2 g, about 1400 mg toabout 5 g, about 1400 mg to about 4 g, about 1400 mg to about 3 g, about1400 mg to about 2 g, about 1600 mg to about 5 g, about 1600 mg to about4 g, about 1600 mg to about 3 g, about 1600 mg to about 2 g, about 1800mg to about 5 g, about 1800 mg to about 4 g, about 1800 mg to about 3 g,or about 1800 mg to about 2 g of the pharmaceutically active ingredient.Certain embodiments include unit doses that contain about 900 mg, about1200 mg, and about 1800 mg of the pharmaceutically active ingredient.

In certain embodiments, the unit dose can contain between about 200 mgand about 900 mg of the pharmaceutically active ingredient of thedisclosure, such as about 200 mg, about 250 mg, about 300 mg, about 350mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850mg, or about 900 mg of the pharmaceutically active ingredient.

Pharmaceutical compositions of the hydroxypropyl beta-cyclodextrinmixtures of the disclosure can be prepared for storage as lyophilizedformulations or aqueous solutions by mixing the pharmaceutically activeingredient having the desired degree of purity with optionalpharmaceutically-acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are referred to herein as“carriers”), i.e., buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants, and othermiscellaneous additives. See, Remington's Pharmaceutical Sciences, 16thedition (Osol, ed. 1980).

In some embodiments, buffering agents are used to help to maintain thepH in the range that approximates physiological conditions from about 2mM to about 50 mM, such as about 2 to about 40, about 2 to about 30,about 2 to about 20, about 2 to about 10, about 10 to about 50, about 10to about 40, about 10 to about 30, about 10 to about 20, about 20 toabout 50, about 20 to about 40, about 20 to about 30, or about 40 toabout 50 mM. For example, one or more buffering agents can be present ata concentration of about 2, about 5, about 10, about 15, about 20, about25, about 30, about 35, about 40, about 45, or about 50 mM. Suitablebuffering agents for use with the present disclosure include bothorganic and inorganic acids and salts thereof, such as citrate buffers(e.g., monosodium citrate-disodium citrate mixture, citricacid-trisodium citrate mixture, citric acid-monosodium citrate mixture,etc.), succinate buffers (e.g., succinic acid-monosodium succinatemixture, succinic acid-sodium hydroxide mixture, succinic acid-disodiumsuccinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodiumtartrate mixture, tartaric acid-potassium tartrate mixture, tartaricacid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium glyconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium glyuconatemixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) and acetate buffers (e.g., aceticacid-sodium acetate mixture, acetic acid-sodium hydroxide mixture,etc.). Additionally, phosphate buffers, histidine buffers andtrimethylamine salts such as Tris can be used.

In some embodiments, preservatives are added to retard microbial growth,in amounts ranging from 0.01%-1% (w/v), such as 0.1%-1%, 0.2%-1%,0.3%-1%, 0.5%-1%, 0.01%-0.5%, 0.02%-0.5%, 0.05%-0.5%, 0.1%-0.5%,0.2%-0.5%, or 0.05%-0.2% (w/v). For example, a preservative in an amountof about 0.02%, about 0.05%, about 0.1%, about 0.2%, about 0.5%, about0.8% (w/v) can be added. Suitable preservatives for use with the presentdisclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben,propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconiumhalides (e.g., chloride, bromide, and iodide), hexamethonium chloride,and alkyl parabens such as methyl or propyl paraben, catechol,resorcinol, cyclohexanol, and 3-pentanol.

In some embodiments, isotonifiers sometimes known as “stabilizers” areadded to ensure isotonicity of liquid compositions of the presentdisclosure and include polyhydric sugar alcohols, for example trihydricor higher sugar alcohols, such as glycerin, erythritol, arabitol,xylitol, sorbitol and mannitol. Stabilizers refer to a broad category ofexcipients which can range in function from a bulking agent to anadditive which solubilizes the therapeutic agent or helps to preventdenaturation or adherence to the container wall. Typical stabilizers canbe polyhydric sugar alcohols (enumerated above); amino acids such asarginine, lysine, glycine, glutamine, asparagine, histidine, alanine,ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.,organic sugars or sugar alcohols, such as lactose, trehalose, stachyose,mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glyceroland the like, including cyclitols such as inositol; polyethylene glycol;amino acid polymers; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,α-monothioglycerol and sodium thio sulfate; low molecular weightpolypeptides (e.g., peptides of 10 residues or fewer); proteins such ashuman serum albumin, bovine serum albumin, gelatin or immunoglobulins;hydrophylic polymers, such as polyvinylpyrrolidone monosaccharides, suchas xylose, mannose, fructose, glucose; disaccharides such as lactose,maltose, sucrose and trisaccacharides such as raffinose; andpolysaccharides such as dextran. In some embodiments, stabilizers arepresent in the range from 0.1 to 10,000 weights per part of weight ofpharmaceutically active ingredient, such as 0.1 to 1,000, 0.2 to 2,000,0.5 to 5,000, 1 to 10,000, or 1 to 1,000 weights per part of weight ofpharmaceutically active ingredient. For example, stabilizers can bepresent in about 0.2, about 0.5, about 1, about 5, about 10, about 20,about 50, about 100, about 200, about 500, about 1,000, about 2,000,about 5,000, or about 8,000 weights per part of weight ofpharmaceutically active ingredient.

In some embodiments, ionic surfactants are added to help solubilize thetherapeutic agent as well as to protect the active ingredient againstagitation-induced aggregation. In some embodiments, non-ionicsurfactants or detergents (also known as “wetting agents”) are added tohelp solubilize the therapeutic agent as well as to protect the activeingredient against agitation-induced aggregation. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). In some embodiments, non-ionic surfactants are presentin a range of from about 0.05 mg/mL to about 1.0 mg/mL, such as about0.05 mg/mL to about 0.2 mg/mL, about 0.07 mg/mL to about 0.2 mg/mL,about 0.1 mg/mL to about 0.3 mg/mL, or about 0.1 mg/mL to about 0.5mg/mL. For instance, non-ionic surfactants can be present in about 0.05,about 0.07, about 0.08, about 0.1, about 0.2, about 0.3, about 0.4,about 0.5, about 0.6, about 0.8, or about 1.0 mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

In some embodiments, the pharmaceutical composition herein also containsa combination of therapeutic agents, e.g., a second therapeutic agent inaddition to the pharmaceutically active ingredient of the disclosure(the mixture of beta-cyclodextrins described herein). Examples ofsuitable combination therapeutic agents are provided in Section 6.3.3.below.

In some embodiments, the pharmaceutical compositions described hereinsolubilize lipids in an aqueous medium. The aqueous medium can be, forexample, distilled water or deionized water, or can be an aqueousenvironment, e.g., blood, cerebrospinal fluid, or lymphatic fluid, inthe body of a subject. The solubilizing ability of the compositions canbe typically measured by UV transmission properties of the solution,e.g., as monitored by UV spectrometry or by HPLC, with a decrease intransmission correlating with formation of a suspension within thesolution. In some embodiments, the lipids that are solubilized compriseunesterified or esterified cholesterol; cholesterol metabolites, e.g.,7-ketocholesterol, 7β-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 27-hydroxycholesterol, andcholestane-3β,5α,6β-triol; sphingolipids; glycolipids; ceramides;gangliosides, e.g., GM2((2S,3R,4E)-3-Hydroxy-2-(octadecanoylamino)octadec-4-en-1-yl2-acetamido-2-deoxy-β-D-galactopyranosyl-(1→4)-[5-acetamido-3,5-dideoxy-D-glycero-α-D-galacto-non-2-ulopyranonosyl-(2→3)]-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside)or GM3 (monosialodihexosylganglioside); or any combination thereof.

As used herein, a weight per volume (“weight/volume” or “w/v”) solutionrefers to the weight of a solute dissolved in a volume of water. In anillustrative example, a 10% (w/v) solution of hydroxypropylbeta-cyclodextrins has 1 g of the solute in 10 mL of the aqueoussolution. In another example, a 20% (w/v) solution of hydroxypropylbeta-cyclodextrins has 200 mg of the solute in 1 mL of the aqueoussolution.

In some embodiments, 1 mL of a 20% (w/v) solution of thepharmaceutically active ingredient described herein solubilizes at least2 mg, such as at least 3 mg, at least 4 mg, at least 5 mg, at least 6mg, at least 7 mg, at least 8 mg, or at least 10 mg, of unesterifiedcholesterol in distilled water at room temperature when measured, e.g.,by UV spectrometry, after about 24 hours. In some embodiments, about 200mg of the pharmaceutically active ingredient solubilizes at least 2 mg,such as at least 3 mg, at least 4 mg, at least 5 mg, at least 6 mg, atleast 7 mg, at least 8 mg, or at least 10 mg of unesterified cholesterolin distilled water at room temperature when measured after about 24hours.

6.3. Methods of Treatment

The disclosure provides a method of treating Niemann-Pick disease Type C(NPC), such as Niemann-Pick disease Type C1 (NPC1) or Niemann-Pickdisease Type C2 (NPC2), comprising administering to a subject havingNiemann-Pick disease a therapeutically effective amount of apharmaceutical composition as described herein.

As used herein, a “subject” is a mammal, such as a mouse, a rat, a cat,a dog, a cow, a pig, a horse. In some embodiments, the mammal is aprimate, such as a monkey, a chimpanzee, or a human. For example, asubject can be a human subject, i.e., a human patient. In certainembodiments, the patient is a pediatric patient or an adult patient.Pediatric human patients include pediatric patients with diseasecharacterized at early-infantile onset (less than 2 years of age),late-infantile onset (2 to less than 6 years of age), juvenile onset (6to less than 15 years of age) and adolescent onset (15 years of age orgreater).

The terms “treat”, “treating” or “treatment” refer to administration ofa pharmaceutical composition described herein so as to modulatebeneficially a level of one or more lipid biomarkers or therapeuticeffects as described in Section 6.3.4. compared to a baseline level. Anexemplary treatment phase involves a repeat administration of apharmaceutical composition described herein where the score of one ormore domains of the NPC Severity Scale as defined in Section 6.3.4.2 isreduced compared to a prior baseline value.

The terms “maintain”, “maintaining” or “maintenance” refer toadministration of a pharmaceutical composition described herein to holdconstant a baseline level of one or more biological effects as describedin Section 6.3.4. A maintenance phase of administration may preventprogression of NPC as compared with no administration or administrationof a placebo. An illustrative example of a maintenance phase is a repeatadministration of a pharmaceutical composition described herein wherethe score of one or more domains of the NPC Severity Scale as defined inSection 6.3.4.2 is held at the same level as a baseline value.

6.3.1. Administration of the Hydroxypropyl Beta-CyclodextrinPharmaceutical Compositions

In some embodiments, the method comprises administering about 200 mg toabout 3000 mg, such as about 200 to about 2800, about 200 to about 2600,about 200 to about 2500, about 200 to about 2400, about 200 to about2200, about 200 to about 2000, about 200 to about 1800, about 200 toabout 1600, about 200 to about 1500, about 200 to about 1200, about 200to about 1100, about 200 to about 1000, about 200 to about 800, about200 to about 700, about 200 to about 600, about 200 to about 500, about200 to about 400, about 200 to about 300; about 300 to about 3000, about300 to about 2800, about 300 to about 2600, about 300 to about 2500,about 300 to about 2400, about 300 to about 2200, about 300 to about2000, about 300 to about 1800, about 300 to about 1600, about 300 toabout 1500, about 300 to about 1200, about 300 to about 1100, about 300to about 1000, about 300 to about 800, about 300 to about 700, about 300to about 600, about 300 to about 500, about 300 to about 400; such asfrom about 400 to about 3000, about 400 to about 2800, about 400 toabout 2600, about 400 to about 2500, about 400 to about 2400, about 400to about 2200, about 400 to about 2000, about 400 to about 1800, about400 to about 1600, about 400 to about 1500, about 400 to about 1200,about 400 to about 1100, about 400 to about 1000, about 400 to about800, about 400 to about 700, about 400 to about 600, about 400 to about500; such as from about 500 to about 3000, about 500 to about 2800,about 500 to about 2600, about 500 to about 2500, about 500 to about2400, about 500 to about 2200, about 500 to about 2000, about 500 toabout 1800, about 500 to about 1600, about 500 to about 1500, about 500to about 1200, about 500 to about 1100, about 500 to about 1000, about500 to about 800, about 500 to about 700, about 500 to about 600; suchas from about 600 to about 3000, about 600 to about 2800, about 600 toabout 2600, about 600 to about 2500, about 600 to about 2400, about 600to about 2200, about 600 to about 2000, about 600 to about 1800, about600 to about 1600, about 600 to about 1500, about 600 to about 1200,about 600 to about 1100, about 600 to about 1000, about 600 to about800, about 600 to about 700; such as from about 700 to about 3000, about700 to about 2800, about 700 to about 2600, about 700 to about 2500,about 700 to about 2400, about 700 to about 2200, about 700 to about2000, about 700 to about 1800, about 700 to about 1600, about 700 toabout 1500, about 700 to about 1200, about 700 to about 1100, about 700to about 1000, about 700 to about 800; such as from about 800 to about3000, about 800 to about 2800, about 800 to about 2600, about 800 toabout 2500, about 800 to about 2400, about 800 to about 2200, about 800to about 2000, about 800 to about 1800, about 800 to about 1600, about800 to about 1500, about 800 to about 1200, about 800 to about 1100,about 800 to about 1000; such as from about 1000 to about 3000, about1000 to about 2800, about 1000 to about 2600, about 1000 to about 2500,about 1000 to about 2400, about 1000 to about 2200, about 1000 to about2000, about 1000 to about 1800, about 1000 to about 1600, about 1000 toabout 1500, about 1000 to about 1200, about 1000 to about 1100: such asfrom about 1100 to about 3000, about 1100 to about 2800, about 1100 toabout 2600, about 1100 to about 2500, about 1100 to about 2400, about1100 to about 2200, about 1100 to about 2000, about 1100 to about 1800,about 1100 to about 1600, about 1100 to about 1500, about 1100 to about1200; such as from about 1200 to about 3000, about 1200 to about 2800,about 1200 to about 2600, about 1200 to about 2500, about 1200 to about2400, about 1200 to about 2200, about 1200 to about 2000, about 1200 toabout 1800, about 1200 to about 1600, about 1200 to about 1500; such asfrom about 1500 to about 3000, about 1500 to about 2800, about 1500 toabout 2600, about 1500 to about 2500, about 1500 to about 2400, about1500 to about 2200, about 1500 to about 2000, about 1500 to about 1800,about 1500 to about 1600; such as from about 1600 to about 3000, about1600 to about 2800, about 1600 to about 2600, about 1600 to about 2500,about 1600 to about 2400, about 1600 to about 2200, about 1600 to about2000, about 1600 to about 1800; such as from about 1800 to about 3000,about 1800 to about 2800, about 1800 to about 2600, about 1800 to about2500, about 1800 to about 2400, about 1800 to about 2200, about 1800 toabout 2000; such as from about 2000 to about 3000, about 2000 to about2800, about 2000 to about 2600, about 2000 to about 2500, about 2000 toabout 2400, about 2000 to about 2200; such as from about 2200 to about3000, about 2200 to about 2800, about 2200 to about 2600, about 2200 toabout 2500, about 2200 to about 2400; such as from about 2400 to about3000, about 2400 to about 2800, about 2400 to about 2600, about 2400 toabout 2500; such as from about 2500 to about 3000, about 2500 to about2800, about 2500 to about 2600; such as from about 2600 to about 3000,about 2600 to about 2800; or about 2800 to about 3000 mg, of thepharmaceutically active ingredient to the subject per administration.

In some embodiments, the dosage schedule consists of administration onceevery week, once every two weeks, once every three weeks, once a month,once every two months, or once every three months. For example, themethod can comprise administering about 200, about 300, about 400, about500, about 600, about 700, about 800, about 900, about 1000, about 1200,about 1400, about 1500, about 1600, about 1800, about 2000, about 2200,about 2400, about 2500, or about 3000 mg of the pharmaceutically activeingredient to the subject per administration.

In some embodiments, the administering occurs in a single dose peradministration. In other embodiments, the pharmaceutical composition isadministered in divided doses, with the overall dose divided into twodoses, three doses, or even four doses, per administration, e.g., over aweek, two weeks, a month, two months, etc., specifically over two weeks.The composition may also be administered continuously, or in anyeffective range or value therein depending on the condition beingtreated, the route of administration and the age, weight and conditionof the subject.

In some embodiments, the pharmaceutical compositions of the disclosureare suitable for intrathecal or intracerebroventricular administration.In certain embodiments, intrathecal administration of the pharmaceuticalcomposition is through an intrathecal port. In certain of theseembodiments, the port is a Celsite® port (B. Braun Medical, France). Incertain embodiments, the intrathecal administration comprisesadministering as an intrathecal slow bolus (1-2 minute, depending on thevolume administered) lumbar puncture injection (maximum rate ofadministration=4.5 mL/minute). In certain embodiments, the techniques oflumbar puncture include use of a non-cutting needle, such as a Whiteacreor Sprotte needle, insertion parallel to dural fibers, and replacingstylet prior to needle removal. In certain embodiments, prior toinjection, a volume of CSF fluid equal to the volume to be administeredis removed.

In another illustrative example, intracerebroventricular administrationcan be through an Ommaya reservoir.

For treatment of NPC described herein, an effective dose of thepharmaceutically active ingredient, the hydroxypropyl beta-cyclodextrinmixture, can range from about 0.001 to about 1000 mg/kg, such as about0.1 to about 1000, about 1 to about 1000, about 10 to about 1000, about20 to about 1000, about 50 to about 1000, about 100 to about 1000, about200 to about 1000, about 300 to about 1000, about 400 to about 1000,about 500 to about 1000, about 600 to about 1000, about 800 to about1000; such as from about 0.1 to about 800, about 1 to about 800, about10 to about 800, about 20 to about 800, about 50 to about 800, about 100to about 800, about 200 to about 800, about 300 to about 800, about 400to about 800, about 500 to about 800, about 600 to about 800; such asfrom about 0.1 to about 600, about 1 to about 600, about 10 to about600, about 20 to about 600, about 50 to about 600, about 100 to about600, about 200 to about 600, about 300 to about 600, about 400 to about600, about 500 to about 600; such as from about 0.1 to about 500, about1 to about 500, about 10 to about 500, about 20 to about 500, about 50to about 500, about 100 to about 500, about 200 to about 500, about 300to about 500, about 400 to about 500; such as from about 0.1 to about400, about 1 to about 400, about 10 to about 400, about 20 to about 400,about 50 to about 400, about 100 to about 400, about 200 to about 400,about 300 to about 400; such as from about 0.1 to about 300, about 1 toabout 300, about 10 to about 300, about 20 to about 300, about 50 toabout 300, about 100 to about 300, about 200 to about 300; such as fromabout 0.1 to about 200, about 1 to about 200, about 10 to about 200,about 20 to about 200, about 50 to about 200, about 100 to about 200;such as from about 0.1 to about 100, about 1 to about 100, about 10 toabout 100, about 20 to about 100, about 50 to about 100; such as fromabout 0.1 to about 50, about 1 to about 50, about 10 to about 50, about20 to about 50; such as from about 0.1 to about 20, about 1 to about 20,about 10 to about 20; such as from about 0.1 to about 10, about 1 toabout 10; or about 0.1 to about 1 mg/kg.

In some embodiments, the method comprises a treatment phase wherein theadministering occurs every week, every two weeks, every three weeks, orevery month, in order to reduce symptoms of NPC.

In some embodiments, the method comprises a maintenance phase whereinthe administering occurs every three weeks, every month, every twomonths, or every three months, in order to maintain a steady state ofthe disease.

In some embodiments, the pharmaceutical composition is administered as abolus, followed by a continuous maintenance dose.

In certain embodiments, the pharmaceutical composition is administeredmonthly through intrathecal or intracerebroventricular administration.In certain embodiments, the pharmaceutical composition is administeredcontinuously through intrathecal or intracerebroventricularadministration.

In some embodiments, the method comprises a treatment phase wherein 900mg of the pharmaceutically active ingredient is administered to thepatient as initial doses and a maintenance phase wherein less than 900mg of the pharmaceutically active ingredient is administered every otherweek by intrathecal administration.

6.3.2. Multiple Routes of Administration

In some embodiments, the method comprises administering thepharmaceutically active ingredient using multiple routes ofadministration. In certain embodiments, the method comprisesadministering the pharmaceutically active ingredient (i) intrathecallyor by intracerebroventricular administration, and (ii) intravenously.These embodiments usefully allow reduction of cholesterol accumulationin both the central nervous system and peripheral organs.

In some embodiments, the intravenous administration comprisesadministering about 200 mg/kg to about 4000 mg/kg of thebeta-cyclodextrin mixture by intravenous infusion over 6 to 8 hours tothe patient. In some embodiments, the intravenous administrationcomprises administering about 500 mg/kg to about 4000 mg/kg of thebeta-cyclodextrin mixture by intravenous infusion over 6 to 8 hours tothe patient.

In typical embodiments, the pharmaceutical composition comprises about200 mg/mL of the beta-cyclodextrin mixture. In certain otherembodiments, the pharmaceutical composition comprises about 250 mg/mL ofthe beta-cyclodextrin mixture. In some embodiments, the pharmaceuticalcomposition is administered once every three days, once every week, onceevery two weeks, once every three weeks, once every month, once everytwo months, or once every three months. In certain embodiments,intravenous administration is started shortly after birth. In certainother embodiments, intravenous administration is started afterintrathecal (or intracerebroventricular) administration is initiated. Insome embodiments, the liver volume, the spleen volume, and/or liverenzyme activity of the patient are monitored to determine the efficacyof the treatment, and for adjustment of dosage schedule.

6.3.3. Combination Therapy

Described below are combination therapy methods in which thehydroxypropyl beta-cyclodextrin pharmaceutical compositions of thedisclosure can optionally be utilized. In some embodiments, thecombination methods of the disclosure involve the administration of atleast two agents to a subject, the first of which is the hydroxypropylbeta-cyclodextrin mixture described herein (for example, in apharmaceutical composition described herein), and the additionalagent(s) is a combination therapeutic agent. The hydroxypropylbeta-cyclodextrin mixture and the combination therapeutic agent(s) canbe administered simultaneously (e.g., in a pharmaceutical composition asdescribed herein), sequentially, or separately.

The combination therapy methods of the present disclosure can result ina greater than additive effect, i.e., a synergistic effect, for example,providing therapeutic benefits greater than the expected sum of thebenefit from the hydroxypropyl beta-cyclodextrin mixture and thecombination therapeutic agent when each is administered individually.

In some embodiments, the hydroxypropyl beta-cyclodextrin mixture and thecombination therapeutic agent are administered concurrently, eithersimultaneously or successively. As used herein, the hydroxypropylbeta-cyclodextrin mixture and the combination therapeutic agent are saidto be administered successively if they are administered to the subjecton the same day, for example, during the same subject visit. Successiveadministration can occur 1, 2, 3, 4, 5, 6, 7, or 8 hours apart. Incontrast, the hydroxypropyl beta-cyclodextrin mixture and thecombination therapeutic agent are said to be administered separately ifthey are administered to the subject on different days, for example, thehydroxypropyl beta-cyclodextrin mixture and the combination therapeuticagent can be administered at a 1-day, 2-day or 3-day, 1-week, 2-week ormonthly intervals. In the methods of the present disclosure,administration of the hydroxypropyl beta-cyclodextrin mixture canprecede or follow administration of the combination therapeutic agent.

As a non-limiting example, the hydroxypropyl beta-cyclodextrin mixtureand the combination therapeutic agent can be administered concurrentlyfor a period of time, followed by a second period of time in which theadministration of the hydroxypropyl beta-cyclodextrin mixture and thecombination therapeutic agent is alternated.

Because of the potentially synergistic effects of administering thehydroxypropyl beta-cyclodextrin mixture and the combination therapeuticagent, such agents can be administered as a therapeutically effectivecombination in amounts that are not therapeutically effective if one orboth of the agents were administered alone.

In certain embodiments, the combination therapeutic agent is a vitamin Eor a derivative thereof, an enzyme replacement therapy, a steroid, aglucosyl transferase enzyme inhibitor, a histone deacetylase (HDAC)inhibitor, or a molecular chaperone activator.

Vitamin E or vitamin E derivatives include but are not limited toalpha-tocopherol, delta-tocopherol, and tocopherol derivatives. In someembodiments, vitamin E derivatives include esterified tocopherols, e.g.,tocopheryl acetate, and chemically related tocopherol derivatives suchas those described in PCT Publication No. WO 2014/078573, incorporatedherein by reference in its entirety.

Enzyme replacement therapies include but are not limited to agalsidasebeta (Fabrazyme®), imiglucerase (Cerezyme®), verlaglucerase alfa(VPRIV™), taliglucerase (Elelyso™), alglucosidase alfa (Myozyme® orLumizyme®), laronidase (Aldurazyme®), idursulfase intravenous(Elaprase®), and galsulfase (Naglazyme™).

Steroids include but are not limited to neurosteroids, such asallopregnanolone and ganaxolone.

Glucosyl transferase enzyme inhibitors include but are not limited toglucoceramide synthase inhibitors, such as miglustat (Zavesca®).

HDAC inhibitors include but are not limited to vironostat, romidepsin,trichostatin A, valproate, butyrate, trapoxins, and apicidin.

The molecular chaperone activators include but are not limited toarimoclomol.

6.3.4. Biological Effects of the Methods

6.3.4.1. Effect on Biomarkers

In some embodiments, the methods of treating NPC comprise maintaining ormodulating levels of one or more lipids, such as unesterifiedcholesterol or gangliosides, that have accumulated in one or more bodyorgans, e.g., in the brain, and that lead to disease symptoms. Incertain embodiments, modulating levels of one or more lipids includeslowering levels of one or more lipids. In some embodiments, the efficacyof the methods is determined by measuring the level of storage of one ormore lipids before (baseline level) and after the start of treatment.For example, in some embodiments, the subject methods lower levels ofone or more lipids by about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%relative to a baseline level.

In some embodiments, the subject methods lower levels of one or morelipids by 20%±5%, 25%±5%, 30%±5%, 35%±5%, 40%±5%, 45%±5%, 50%±5%,55%±5%, 60%±5%, 65%±5%, 70%±5%, 75%±5%, 80%±5%, 85%±5%, 90%±5%, or95%±5%, relative to a baseline level.

In some embodiments, the subject methods lower levels of one or morelipids by 20%±3%, 25%±3%, 30%±3%, 35%±3%, 40%±3%, 45%±3%, 50%±3%,55%±3%, 60%±3%, 65%±3%, 70%±3%, 75%±3%, 80%±3%, 85%±3%, 90%±3%, 95%±3%,or 97%±3%, relative to a baseline level.

In some embodiments, the subject methods lower levels of one or morelipids by 20%±2%, 25%±2%, 30%±2%, 35%±2%, 40%±2%, 45%±2%, 50%±2%,55%±2%, 60%±2%, 65%±2%, 70%±2%, 75%±2%, 80%±2%, 85%±2%, 90%±2%, 95%±2%,or 97%±2%, relative to a baseline level.

In some embodiments, the subject methods lower levels of one or morelipids by at least about 20%, such as at least about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 97%, relative to a baseline level.

In some embodiments, the subject methods lower levels of one or morelipids in a range of from about 20% to about 97%, such as about 20% toabout 95%, about 20% to about 90%, about 20% to about 85%, about 20% toabout 80%, about 20% to about 75%, about 20% to about 70%, about 20% toabout 65%, about 20% to about 60%, about 20% to about 55%, about 20% toabout 50%, about 20% to about 45%, about 20% to about 40%, about 20% toabout 35%, about 20% to about 30%, about 20% to about 25%; such as fromabout 25% to about 97%, about 25% to about 95%, about 25% to about 90%,about 25% to about 85%, about 25% to about 80%, about 25% to about 75%,about 25% to about 70%, about 25% to about 65%, about 25% to about 60%,about 25% to about 55%, about 25% to about 50%, about 25% to about 45%,about 25% to about 40%, about 25% to about 35%, about 25% to about 30%;such as from about 30% to about 97%, about 30% to about 95%, about 30%to about 90%, about 30% to about 85%, about 30% to about 80%, about 30%to about 75%, about 30% to about 70%, about 30% to about 65%, about 30%to about 60%, about 30% to about 55%, about 30% to about 50%, about 30%to about 45%, about 30% to about 40%, about 30% to about 35%; such asfrom about 35% to about 97%, about 35% to about 95%, about 35% to about90%, about 35% to about 85%, about 35% to about 80%, about 35% to about75%, about 35% to about 70%, about 35% to about 65%, about 35% to about60%, about 35% to about 55%, about 35% to about 50%, about 35% to about45%, about 35% to about 40%; such as from about 40% to about 97%, about40% to about 95%, about 40% to about 90%, about 40% to about 85%, about40% to about 80%, about 40% to about 75%, about 40% to about 70%, about40% to about 65%, about 40% to about 60%, about 40% to about 55%, about40% to about 50%, about 40% to about 45%; such as from about 45% toabout 97%, about 45% to about 95%, about 45% to about 90%, about 45% toabout 85%, about 45% to about 80%, about 45% to about 75%, about 45% toabout 70%, about 45% to about 65%, about 45% to about 60%, about 45% toabout 55%, about 45% to about 50%; such as from about 50% to about 97%,about 50% to about 95%, about 50% to about 90%, about 50% to about 85%,about 50% to about 80%, about 50% to about 75%, about 50% to about 70%,about 50% to about 65%, about 50% to about 60%, about 50% to about 55%;such as from about 55% to about 97%, about 55% to about 95%, about 55%to about 90%, about 55% to about 85%, about 55% to about 80%, about 55%to about 75%, about 55% to about 70%, about 55% to about 65%, about 55%to about 60%; such as from about 60% to about 97%, about 60% to about95%, about 60% to about 90%, about 60% to about 85%, about 60% to about80%, about 60% to about 75%, about 60% to about 70%, about 60% to about65%; such as from about 65% to about 97%, about 65% to about 95%, about65% to about 90%, about 65% to about 85%, about 65% to about 80%, about65% to about 75%, about 65% to about 70%; such as from about 70% toabout 97%, about 70% to about 95%, about 70% to about 90%, about 70% toabout 85%, about 70% to about 80%, about 70% to about 75%; such as fromabout 75% to about 97%, about 75% to about 95%, about 75% to about 90%,about 75% to about 85%, about 75% to about 80%; such as from about 80%to about 97%, about 80% to about 95%, about 80% to about 90%, about 80%to about 85%; such as from about 85% to about 97%, about 85% to about95%, about 85% to about 90%; such as from about 90% to about 97%, about90% to about 95%, or about 95% to about 97%, relative to a baselinelevel.

In an NPC patient, measuring the level of storage of one or more lipidscan be performed by monitoring one or more biomarkers in a sample ofcerebrospinal fluid (CSF), plasma, or urine. In some embodiments, CSF isused to determine the excretion levels of the one or more lipidsdirectly. In some embodiments, a downstream protein biomarker that hasbeen modulated by the change in the levels of one or more lipids ismonitored. In some embodiments, the method comprises administering anamount of the pharmaceutically active ingredient sufficient to modulate,e.g., lower relative to a baseline level, the level in cerebrospinalfluid of one or more of: tau protein, amyloid peptide, neurofilamentlight protein (NFL), glial fibrillary acidic protein (GFAP), sterol,oxysterol, chitotriosidase activity, calbindin, lysosomal-associatedmembrane protein 1 (LAMP-1), GM2 or GM3 ganglioside, sphingosine, andsphingosine-1-phosphate (SIP).

In some embodiments, plasma samples are used to determine the levels ofone or more lipids, e.g., cholesterol or cholesterol metabolites,present in the blood before and after the start of treatment. In someembodiments, the method comprises administering an amount of thepharmaceutically active ingredient sufficient to modulate, e.g., lowerrelative to a baseline level, the level in plasma of one or more of:7-ketocholesterol, 7β-hydroxycholesterol, 24S-hydroxycholesterol,25-hydroxycholesterol, 27-hydroxycholesterol, andcholestane-3β,5α,6β-triol.

Monitoring other lipids, such as metabolites like3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CA),glycine-conjugated 3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oicacid (SNAG-Δ⁵-CG), and taurine-conjugated3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CT),that have been reported in the urine of NPC1 patients may provide usefulbiomarkers (Maekawa, M. et al. “Focused metabolomics using liquidchromatography/electrospray ionization tandem mass spectrometry foranalysis of urinary conjugated cholesterol metabolites from patientswith Niemann-Pick disease type C and 3β-hydroxysteroid dehydrogenasedeficiency.” Annals of Clinical Biochemistry OnlineFirst, published Mar.2, 2015). In some embodiments, the method comprises administering anamount of the pharmaceutically active ingredient sufficient to modulate,e.g., lower relative to a baseline level, the level in urine of one ormore of: 3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid(SNAG-Δ⁵-CA), glycine-conjugated3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CG),and taurine-conjugated3β-sulfoxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid (SNAG-Δ⁵-CT).

6.3.4.2. Therapeutic Effects

In some embodiments, the methods of the disclosure have a beneficialeffect on one or more symptoms of NPC.

WWI One measure to characterize and quantify NPC disease progression isthrough the use of the NPC Severity Scale, which determines clinicalsigns and symptoms in nine major domains (ambulation, cognition, eyemovement, fine motor skills, hearing, memory, seizures, speech, andswallowing) and eight minor domains (auditory brainstem response,behavior, gelastic cataplexy, hyperreflexia, incontinence, narcolepsy,psychiatric, and respiratory problems) (Yanjanin et al., “LinearClinical Progression, Independent of Age of Onset, in Niemann-PickDisease, Type C,” Am. J. Med. Genet. Part B 153B:132-140 (2010))(“Yanjanin 2010”). The overall clinical severity score (or “overallscore”) is the aggregate of all the assessments in each of the major andminor domains, and is determined by the sum of all the individual domainscores See Table 1, below; see also FIG. 1.

TABLE 1 NPC Clinical Severity Scale (from Yanjanin, 2010) Eye MovementScore Ambulation Score Normal eye movement 0 Normal 0 Mild verticalsupranuclear gaze palsy 1 Clumsy 1 (VSGP) detected by physician onlyAtaxic unassisted gait or not 2 walking by 18 months Functional VSGP,noted by family or 2 Assisted ambulation or not 4 compensation with headmovements walking by 24 months Total VSGP, abnormal horizontal 3Wheelchair dependent 5 saccades may be present Total ophthalmoplegia(vertical and 5 horizontal saccades absent) Speech Score Swallow ScoreNormal speech 0 Normal, no dysphagia 0 Mild dysarthria (easilyunderstood) 1 Cough while eating 1 Severe dysarthria (difficult to 2Intermittent dysphagia* w/Liquids +1 understand) w/Solids +1Non-verbal/functional communication 3 Dysphagia* w/Liquids +2 skills forneeds w/Solids +2 Minimal communication 5 Nasogastric tube or gastrictube 4 for supplemental feeding Nasogastric tube or gastric tube 5feeding only Fine Motor Skills Score Cognition Score Normal 0 Normal 0Slight dysmetria/dystonia 1 Mild learning delay, grade 1 (independentmanipulation) appropriate for age Mild dysmetria/Dystonia (requires 2Moderate learning delay, 3 little to no assistance, able toindividualized curriculum or feed self without difficulty) modified worksetting Moderate dysmetria/dystonia (limited 4 Severe delay/plateau, nolonger in 4 fine motor skills, difficulty feeding school or no longerable to work, self) some loss of cognitive function SevereDysmetria/Dystonia (gross 5 Minimal cognitive function 5 motorlimitation, requires assistance for self-care activities) Hearing(sensineural) Score Memory Score Normal hearing (all tones ≤15 dB HL) 0Normal 0 High frequency hearing loss (PTA** ≤15 1 Mild short-term orlong-term 1 dB HL, >15 dB HL in high frequencies) memory lossSlight-mild hearing loss (PTA 16-44 2 Moderate short-term or long-term 2dB HL) memory loss (gets lost) Moderate hearing loss (PTA 45-70 dB HL) 3Difficulty following commands 3 Severe hearing loss (PTA 71-90 dB HL) 4Unable to follow commands or short- 4 and long- term memory lossProfound hearing loss (PTA >90 dB HL) 5 Minimal memory 5 Seizures ScoreNo history of seizures 0 Hx of single seizure 1 Rare seizures 2Seizures, well controlled with meds 3 Seizures, difficult to controlwith meds 5 Modifiers Score Gelastic cataplexy No history 0 Definitivehistory +1 Frequent (every month) +2 Narcolepsy No history 0 Definitivehistory +1 Frequent (every month) +2 Behavior No problems 0 Hx of ADHD,aggressive +1 Harmful to self/others +2 Psychiatric No problems 0 Hx ofmild depression +1 Hx of major depression, +2 hallucinations, orpsychotic episodes Hyperreflexia None 0 Mild (3+) +1 Severe (+clonus) +2Incontinence No problems 0 Occasional +1 Frequent +2 Auditory BrainstemResponse (ABR) Normal 0 Abnormal +1 Absent +2 Respiratory No problems 0Hx pneumonia +1 Pneumonia >2x/year or active +2 therapeutic intervention*Score is additive within these two subsections **PTA = pure toneaverage-reported on the audiogram

In some embodiments, the method comprises maintaining or reducing one ormore domain scores of the NPC Severity Scale selected from: ambulation,fine motor skills, cognition, speech, swallowing, eye movement, memory,hearing, seizures, auditory brainstem response, behavior, gelasticcataplexy, hyperreflexia, incontinence, narcolepsy, psychiatric, andrespiratory problems. In some embodiments, the method comprisesmaintaining or reducing one or more domain scores of the NPC SeverityScale selected from: ambulation, fine motor skills, cognition, speech,swallowing, eye movement, memory, hearing, and seizures. In someembodiments, the method comprises maintaining or reducing one or moredomain scores of NPC Severity Scale selected from: ambulation, finemotor skills, cognition, speech, swallowing, memory, and seizures. Insome embodiments, the method comprises maintaining or reducing one ormore domain scores of the NPC Severity Scale selected from: ambulation,fine motor skills, cognition, and swallowing.

In some embodiments, treatment that improves the condition of a patientcomprises reducing the score of one or more domains of the NPC SeverityScale compared to a baseline score. In some embodiments, the reductionin the score ranges from about 20% to about 95%, such as about 30% toabout 95%, about 40% to about 95%, about 50% to about 95%, about 60% toabout 95%, about 70% to about 95%, about 80% to about 95%, about 90% toabout 95%; such as from about 30% to about 90%, about 40% to about 90%,about 50% to about 90%, about 60% to about 90%, about 70% to about 90%,about 80% to about 90%; such as from about 20% to about 80%, about 30%to about 80%, about 40% to about 80%, about 50% to about 80%, about 60%to about 80%, about 70% to about 80%; such as from about 20% to about70%, about 30% to about 70%, about 40% to about 70%, about 50% to about70%, about 60% to about 70%; such as from about 20% to about 60%, about30% to about 60%, about 40% to about 60%, about 50% to about 60%; suchas from about 20% to about 50%, about 30% to about 50%, about 40% toabout 50%; such as from about 20% to about 40%, about 30% to about 40%;or about 20% to about 30%, compared to a baseline score. For example,the reduction in the score can be about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, or about 95%, comparedto a baseline score.

In some embodiments, the subject methods reduce the score of one or moredomains by 20%±5%, 25%±5%, 30%±5%, 35%±5%, 40%±5%, 45%±5%, 50%±5%,55%±5%, 60%±5%, 65%±5%, 70%±5%, 75%±5%, 80%±5%, 85%±5%, 90%±5%, or95%±5%, relative to a baseline level.

In some embodiments, the subject methods reduce the score of one or moredomains by 20%±3%, 25%±3%, 30%±3%, 35%±3%, 40%±3%, 45%±3%, 50%±3%,55%±3%, 60%±3%, 65%±3%, 70%±3%, 75%±3%, 80%±3%, 85%±3%, 90%±3%, 95%±3%,or 97%±3%, relative to a baseline level.

In some embodiments, the subject methods reduce the score of one or moredomains by 20%±2%, 25%±2%, 30%±2%, 35%±2%, 40%±2%, 45%±2%, 50%±2%,55%±2%, 60%±2%, 65%±2%, 70%±2%, 75%±2%, 80%±2%, 85%±2%, 90%±2%, 95%±2%,or 97%±2%, relative to a baseline level.

In some embodiments, treatment that improves the condition of a patientcomprises reducing the overall score of the NPC Severity Scale ascompared to a baseline overall score. In some embodiments, the reductionin the overall score ranges from about 20% to about 97%, such as about25% to about 97%, about 30% to about 97%, about 35% to about 97%, about40% to about 97%, about 45% to about 97%, about 50% to about 97%, about55% to about 97%, about 60% to about 97%, about 65% to about 97%, about70% to about 97%, about 75% to about 97%, about 80% to about 97%, about85% to about 97%, about 90% to about 97%; such as from about 20% toabout 95%, about 25% to about 95%, about 30% to about 95%, about 35% toabout 95%, about 40% to about 95%, about 45% to about 95%, about 50% toabout 95%, about 55% to about 95%, about 60% to about 95%, about 65% toabout 95%, about 70% to about 95%, about 75% to about 95%, about 80% toabout 95%, about 85% to about 95%, about 90% to about 95%; such as fromabout 20% to about 90%, about 25% to about 90%, about 30% to about 90%,about 35% to about 90%, about 40% to about 90%, about 45% to about 90%,about 50% to about 90%, about 55% to about 90%, about 60% to about 90%,about 65% to about 90%, about 70% to about 90%, about 75% to about 90%,about 80% to about 90%, about 85% to about 90%; such as from about 20%to about 85%, about 25% to about 85%, about 30% to about 85%, about 35%to about 85%, about 40% to about 85%, about 45% to about 85%, about 50%to about 85%, about 55% to about 85%, about 60% to about 85%, about 65%to about 85%, about 70% to about 85%, about 75% to about 85%, about 80%to about 85%; such as from about 20% to about 80%, about 25% to about80%, about 30% to about 80%, about 35% to about 80%, about 40% to about80%, about 45% to about 80%, about 50% to about 80%, about 55% to about80%, about 60% to about 80%, about 65% to about 80%, about 70% to about80%, about 75% to about 80%; such as from about 20% to about 75%, about25% to about 75%, about 30% to about 75%, about 35% to about 75%, about40% to about 75%, about 45% to about 75%, about 50% to about 75%, about55% to about 75%, about 60% to about 75%, about 65% to about 75%, about70% to about 75%; such as from about 20% to about 70%, about 25% toabout 70%, about 30% to about 70%, about 35% to about 70%, about 40% toabout 70%, about 45% to about 70%, about 50% to about 70%, about 55% toabout 70%, about 60% to about 70%, about 65% to about 70%; such as fromabout 20% to about 65%, about 25% to about 65%, about 30% to about 65%,about 35% to about 65%, about 40% to about 65%, about 45% to about 65%,about 50% to about 65%, about 55% to about 65%, about 60% to about 65%;such as from about 20% to about 60%, about 25% to about 60%, about 30%to about 60%, about 35% to about 60%, about 40% to about 60%, about 45%to about 60%, about 50% to about 60%, about 55% to about 60%; such asfrom about 20% to about 55%, about 25% to about 55%, about 30% to about55%, about 35% to about 55%, about 40% to about 55%, about 45% to about55%, about 50% to about 55%; such as from about 20% to about 50%, about25% to about 50%, about 30% to about 50%, about 35% to about 50%, about40% to about 50%, about 45% to about 50%; such as from about 20% toabout 45%, about 25% to about 45%, about 30% to about 45%, about 35% toabout 45%, about 40% to about 45%; such as from about 20% to about 40%,about 25% to about 40%, about 30% to about 40%, about 35% to about 40%;such as from about 20% to about 35%, about 25% to about 35%, about 30%to about 35%; such as from about 20% to about 30%, about 25% to about30%; or such as from about 20% to about 25%, compared to a baselineoverall score. For example, the reduction in overall score of the NPCSeverity Scale can be about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 97%,compared to a baseline overall score.

In some embodiments, the subject methods reduce the overall score by20%±5%, 25%±5%, 30%±5%, 35%±5%, 40%±5%, 45%±5%, 50%±5%, 55%±5%, 60%±5%,65%±5%, 70%±5%, 75%±5%, 80%±5%, 85%±5%, 90%±5%, or 95%±5%, relative to abaseline overall level.

In some embodiments, the subject methods reduce the overall score by20%±3%, 25%±3%, 30%±3%, 35%±3%, 40%±3%, 45%±3%, 50%±3%, 55%±3%, 60%±3%,65%±3%, 70%±3%, 75%±3%, 80%±3%, 85%±3%, 90%±3%, 95%±3%, or 97%±3%,relative to a baseline overall level.

In some embodiments, the subject methods reduce the overall score by20%±2%, 25%±2%, 30%±2%, 35%±2%, 40%±2%, 45%±2%, 50%±2%, 55%±2%, 60%±2%,65%±2%, 70%±2%, 75%±2%, 80%±2%, 85%±2%, 90%±2%, 95%±2%, or 97%±2%,relative to a baseline overall level.

In some embodiments, a maintenance phase that holds constant thecondition of a patient comprises holding a score of a domain of the NPCSeverity Scale within a range compared to a baseline score. In someembodiments, the maintenance in the score refers to a score within about15%, such as within about 10% or about 5% of the score at a baselinelevel.

In some embodiments, the subject methods result in a score that iswithin 5%±3%, 10%±3%, or 15%±3%, relative to a baseline score. In someembodiments, the subject methods result in a score that is within 5%±2%,10%±2%, or 15%±2%, relative to a baseline score.

In some embodiments, a maintenance phase that holds constant thecondition of a patient comprises holding an overall score of NPCSeverity Scale within a range compared to a baseline overall score. Insome embodiments, the maintenance in the overall score refers to anoverall score within about 15%, such as within about 10% or about 5% ofan overall score at a baseline level.

In some embodiments, the subject methods result in an overall score thatis within 5%±3%, 10%±3%, or 15%±3%, relative to a baseline overallscore. In some embodiments, the subject methods result in an overallscore that is within 5%±2%, 10%±2%, or 15%±2%, relative to a baselineoverall score.

Other measures that can be used to characterize the efficacy of themethods of the disclosure include a global domain comprising a blindedClinician clinical global impression of change (CGI-Clinician) or aCaregiver clinical global impression of change (CGI-Caregiver) followingtreatment, a Timed Up and Go (TUG) test, 9-hole peg test (9-HPT), andquality-of-life measures, such as a United States National Institutes ofHealth PROMIS-PRO (Patient Reported Outcomes Measurement InformationSystem—Patient Reported Outcomes) Caregiver Quality of Life rating.

The methods of the disclosure can be characterized by clinical safetymeasures, which includes one or more of: characterization and severityof clinical adverse events; audiologic testing, e.g., by BAER testing;clinical laboratory tests, e.g., hematology, clinical chemistry,coagulation, urinalysis, CSF analysis; vital signs; physical andneurological exam findings; and electrocardiograms.

6.3.5. Treatment of Other Lysosomal Storage Disorders

As demonstrated in Example 8 (see Section 7.8.2.5), the hydroxypropylbeta-cyclodextrin mixtures described herein have significant effects ongenes related to autophagy, demonstrating that the pharmaceuticallyactive ingredient and pharmaceutical compositions described herein willbe effective in ameliorating certain consequences of the defects inother lysosomal storage disorders.

Accordingly, in another aspect, methods are presented for treatinglysosomal storage disorders other than Niemann-Pick Disease Type C(NPC), comprising administering to a subject having a lysosomal storagedisorder other than NPC a therapeutically effective amount of apharmaceutical composition as described herein.

In various embodiments, the lysosomal storage disorder is selected fromAspartylglucosaminuria, Wolman disease, Cystinosis, Danon disease, Fabrydisease, Farber disease, Fucosidosis, Gaucher disease,GM1-Gangliosidosis types I/II/III, GM2-Gangliosidosis,alpha-Mannosidosis types I/II, beta-Mannosidosis, Metachromaticleukodystrophy, Sialidosis types I/II, Mucolipidosis type IV, Scheiesyndrome, Hunter syndrome, Sanfilippo syndrome A, Sanfilippo syndrome B,Sanfilippo syndrome C, Sanfilippo syndrome D, Galactosialidosis typesI/II, Krabbe disease, Sandhoff disease, Vogt-Spielmeyer disease, Hurlersyndrome, Niemann-Pick disease other than Niemann-Pick Type C, I-celldisease (mucolipidosis II), pseudo-Hurler polydystrophy, Morquiosyndrome, Maroteaux-Lamy syndrome, Sly syndrome, Mucopolysaccharidosistype IX, Multiple sulfatase deficiency, Batten disease, Tay-Sachsdisease, Pompe disease, Batten disease, Batten disease, late infantile,Northern Epilepsy, Pycnodysostosis, Schindler disease, Sialuria, andSalla disease.

In certain embodiments, the lysosomal storage disorder is Tay-Sachsdisease, Sphingolipidoses, Gaucher disease, Mucolipidosis,Galactosialidosis, Salla disorder, Cystinosis, Danon disease, Fabrydisease, Farber disease, Lipofuscinoses, Pompe disease, Gangliodisosis,ISSD, Krabbe disease, Niemann-Pick disease other than NPC,leukodystrophy, Hurler disease, Scheie disease, Hunter disease, SanFilippo disease, Sandhoff disease, Schinder disease, Batten disorder, orWolman disease.

In a further embodiment, the lysosomal storage disorder is Niemann-Pickdisease other than NPC, Tay-Sachs disease, Fabry disease, Farberdisease, San Filippo disease, Batten disorder, or Wolman disease.

EXAMPLES

The following examples are provided by way of exemplification andillustration, not limitation.

7.1. Example 1: Phase I Clinical Trial for Niemann-Pick Disease Type C

A Phase 1 clinical trial was initiated by NIH using a commerciallyavailable parenteral grade hydroxypropyl beta-cyclodextrin mixtureaccording to the following protocol.

7.1.1. Protocol

In this Phase 1, non-randomized, open-label, single-center studyconducted by the NIH, hydroxypropyl beta-cyclodextrin (Kleptose® HPB,Roquette) is administered intrathecally via lumbar injection todrug-naive cohorts of 3 patients each at initial doses of 200 mg,followed by escalation to 300, 400 mg, and 900 mg. All patients in thecohort (three patients per cohort) receive HP-Beta-CD once monthly forat least two doses, and the decision to dose-escalate is based on safetyand on biochemical data. Subsequent dose escalations are effected inincrements of up to 300 mg. Safety is assessed by adverse events (AEs),audiologic evaluation, clinical laboratory tests, vital signs, physicalexaminations, chest X-rays and electrocardiograms (ECGs). Biochemicalefficacy is measured by change from baseline in plasma 24(S)-HC. PK isassessed for plasma HP-Beta-CD.

7.1.2. Drug Product (Kleptose® HPB)

The hydroxypropyl beta-cyclodextrin product used in this Phase IClinical Trial was Kleptose® HPB (Roquette, France) with a DS_(a) ofabout 4.34±10%.

7.1.3. Inclusion Criteria

Patient eligibility inclusion criteria were:

-   -   1) Aged greater than or equal to 2 and less than or equal to 25        years old at time of enrollment, either gender and any        ethnicity.    -   2) Diagnosis of NPC1 based upon one of the following:    -   a) Two NPC1 mutations;    -   b) Positive filipin staining and at least one NPC1 mutation;    -   c) Vertical supranuclear gaze palsy (VSNGP) in combination with        either:    -   i) One NPC1 mutation, or    -   ii) Positive filipin staining and no NPC2 mutations.    -   3) Patients with at least one neurological manifestation of        NPC1. For example, but not limited to, hearing loss, vertical        supranuclear gaze palsy, ataxia, dementia, dystonia, seizures,        dysarthria, or dysphagia.    -   4) Ability to travel to the NIH CC repeatedly for evaluation and        follow-up.    -   5) If taking miglustat, the patient must have been taking a        constant dose of the medication for no less than 3 months prior        to baseline evaluation and must be willing to maintain that dose        level for the duration of the trial.    -   6) Willing to discontinue all non-prescription supplements, with        the exception of an age-appropriate multivitamin.    -   7) Women of reproductive age must be willing to use an effective        method of contraception for the duration of the trial.    -   8) Willing to participate in all aspects of trial design        including serial blood and CSF collections.

7.1.4. Exclusion Criteria

Patient eligibility exclusion criteria were:

-   -   1) Aged below 2 or above 25 years of age at enrollment in the        trial.    -   2) Subjects will be excluded if their weight would result in an        endotoxin level that would exceed 0.2 EU/kg for either the        saline or drug dosing.    -   3) Severe manifestations of NPC1 that would interfere with the        patient s ability to comply with the requirements of this        protocol.    -   4) Neurologically asymptomatic patients.    -   5) Patients who have received any form of cyclodextrin in an        attempt to treat NPC1. Treatment with another drug preparation        for another medical indication that contains cyclodextrin as an        excipient, will not exclude a patient.    -   6) History of hypersensitivity reactions to cyclodextrin or        components of the formulation.    -   7) Pregnancy or breastfeeding at any time during the study.    -   8) Patients with suspected infection of the CNS or any systemic        infection.    -   9) Spinal deformity that would impact the ability to perform a        lumbar puncture.    -   10) Skin infection in the lumbar region.    -   11) Neutropenia, defined as an absolute neutrophil count (ANC)        of less than 1,500.    -   12) Thrombocytopenia (a platelet count of less than 75,000 per        cubic millimeter).    -   13) Evidence of disturbed circulation of CSF.    -   14) Contraindication for anesthesia.    -   15) Prior use of anticoagulants or history/presence of a        bleeding disorder with increased risk of clinical bleeding or an        INR greater than 2.    -   16) Patients with clinical evidence of acute liver disease        having symptoms of jaundice or right upper quadrant pain.    -   17) Presence of anemia defined as two standard deviations below        normal for age and gender.    -   18) For subjects 18 years of age and older, the eGFR is        automatically calculated and reported by the NIH CC laboratory        utilizing the CKD-EPI Creatinine 2009 equation. Subjects greater        than or equal to 18 years of age if eGFR is less than or equal        to 60 mL/min/1.73 m2 are excluded. For subjects <18 years of        age, the NKDEP calculator is utilized        (http://www.nkdep.nih.gov/lab-evaluation/gfr-calculators/children-conventional-unit.shtml).        Results are reported as >75 mL/min/1.73 m2 or lower. Subjects        <18 years of age if eGFR is less than or equal to 75 15        mL/min/1.73 m2 are excluded.    -   19) Hematuria on a single urinalysis, as defined by the American        Urological Association (AUA) as five or more red blood cells per        high-power field (or >25/micro L) on microscopic evaluation of        urinary sediment from a properly collected urinalysis specimen.        The patient will not be excluded if 2 subsequent urine specimens        are negative for hematuria as defined by the AUA.    -   20) Proteinuria (1+protein on urinalysis) unless evaluated and        classified as benign by patient s primary medical provider or by        NIH nephrology consult or in the context of normal urine protein        creatinine ratio and in the absence of clinical symptoms (edema,        hypertension).    -   21) Active pulmonary disease, oxygen requirement or clinically        significant history of decreased blood oxygen saturation,        pulmonary therapy, or requiring active suction.    -   22) Patients unable to complete a behavioral audiologic        evaluation including pure-tone threshold assessment (500 Hz to        8000 Hz) to monitor for ototoxicity and for whom OAEs cannot be        reliably obtained at baseline.    -   23) Patients with ongoing seizures, that are not stable in        frequency, type or duration over a 2 month period prior to        enrollment, requiring change in dose of antiepileptic medication        (other than adjustment for weight) over a 2 month period prior        to enrollment, or requiring 3 or more antiepileptic medications        to control seizures.    -   24) Patients, who in the opinion of the investigators are unable        to comply with the protocol or have specific health concerns        that would potentially increase the risk of participation.

7.1.5. Initial Analysis of Clinical Data

Initial data from this study, with additional data from Individual INDsat another institution also using intrathecal administration ofKleptose® HPB (“I-IND”), were analyzed as follows:

7.1.5.1. Summary of Analysis of Initial Data

We performed an analysis to examine the rate of change in the NPCclinical severity score and its major domains in a data set thatincluded both the NIH subjects and three subjects from an I-IND study atanother institution. The major findings are listed below:

When comparing Table 2 and Table 3 (see below), the drug dose impactsthe rate of change in the NPC clinical severity score and its domains,with the most profound change observed in hearing.

Cyclodextrin generally decreases the rate of decline in the NPC clinicalseverity score and its components (Table 4) (below). This is true forall components with the exception of eye movement, hearing, andseizures.

When limiting the comparison of cyclodextrin to the NIH Natural Historystudy to NIH subjects (Table 5), the pattern is also consistent with theCyclodextrin group declining at a slower rate, with the exception of eyemovement.

The results from the initial analysis of Phase I clinical trial data aresummarized in FIGS. 2-3.

7.1.5.2. Analysis Methods

The goal of the analyses below was to understand the changes over timein the twelve NIH Phase 1 study subjects (identification beginning with“CDA”) and three subjects from another site who are also receivingintrathecal treatment with Kleptose® HPB (identification beginning with“I-IND”). The following results are based on 15 subjects who receivedhydroxypropyl beta-cyclodextrin. Subjects CDA113 and CDA114 are notincluded in the following analyses because they have not yet received adose of hydroxypropyl beta-cyclodextrin. Four (4) out of the 15 subjectshave available data at baseline and 6 months (subjects CDA110, CDA111,CDA112, and I-IND-3); the remaining 11 subjects have available data atbaseline, 6 months, and 12 months (CDA101-CDA109, I-IND-1).

We used a linear mixed model to obtain the estimated slope of changesover time. The word mixed in “linear mixed model” is used to denote thefact that the model includes both “fixed” and “random” effects. The“random” portion of the model accounts for the repeated measures on eachsubject, or longitudinal nature of the data. The “fixed” portion of themodel provides an estimate of the average change over time, that is, theslope of the change. This approach is advantageous because it utilizesall collected data and the interaction term allows for the explorationof different rates of change in groups. This model is fit assuming anunstructured correlation matrix, which means that no assumptions weremade about the correlation between the multiple measurements and eachcorrelation is estimated. In future analyses, covariates may be added tothe models below to explore the effects of covariates such as dose.

We computed regression diagnostics for each model presented in thisanalysis and used Cook's D to identify potential outlier/influentialobservations, refitting models where outliers/influential observationswere identified (Note: an outlier/influential observation is defined asan observation with a Cook's D value of 0.4 or greater). We chose Cook'sD as the diagnostic of interest because it includes both the outcome andany covariates to identify potential outliers. When outliers areidentified, two sets of models are presented; the first model presentedis based on the full data set, while a second set of models is based onthe data with the outliers removed. For this analysis we removed all ofthe I-IND-1 subject's values rather than just the baseline value. Thesevalues are present in the last two columns of Table 2 and Table 3. Notethat the results obtained from these two analyses can be quite differentdue to the relatively small sample size of 15 subjects in the Phase 1study and the total of 41 observations included in the models (when thefull data set is included).

We also used the same approach for the comparison of the Phase 1subjects to the NIH Natural History data set. The NIH Natural Historydata set is described in Yanjanin et al., “Linear Clinical Progression,Independent of Age of Onset, in Niemann-Pick Disease, Type C,” Am. J.Med. Genet. Part B 153B:132-140 (2010).

For the analyses presented here, we fit basic models to the subjects whoreceived cyclodextrin (presented in Table 2). For this analysis, wepresent the estimated change in the corresponding NPC outcome over aperiod of one year. In Table 3, we present results for the averagechange over time while controlling for the dose received. Table 4 andTable 5 present models that compare Cyclodextrin subjects to subjects ina comparable age range in the NIH Natural History study. The resultspresented are the estimated slope in each of the two groups and thep-value for testing the equality of the slopes between the two groups.

7.1.5.3. Changes Over Time in Subjects Receiving Cyclodextrin

Table 2 presents the average slope for the overall NPC score and each ofits components for the 15 subjects who received at least one dose ofcyclodextrin. These results provide information on the rate of change inthe outcome over one year. These rates are smaller than those observedin the full Natural History population that was presented in prioranalyses. Note: we removed the baseline data for one I-IND subject whohad a total score of 31, a total score of 31 with hearing removed,ambulation of 4, swallow of 3, fine motor of 4, and cognition of 4.Overall, this subject improved over time. Additionally, one NIH subjectwas an outlier in the eye movement analysis due to a high value of 5 inthis domain. The analyses for the full data set are presented in columns2 and 3 and the results obtained when outliers were removed arepresented in columns 4 and 5.

TABLE 2 Average change over time (in years) for the NPC score and itscomponents fit to subjects in the Phase 1 studies Avg change Avg changeover time in yrs over time in yrs with I-IND-1 subject removed NPC score(std. error) p-value (std error) p-value Total Score 0.74 (0.59) 0.221.16 (0.42) 0.01 Total Score with 0.31 (0.62) 0.62 0.89 (0.44) 0.06hearing removed Total Score with −0.02 (0.64)  0.97 0.61 (0.46) 0.19hearing and ABR removed^(a) Ambulation 0.001 (0.16) 0.99 0.19 (0.10)0.07 Fine Motor 0.002 (0.08) 0.98 0.002 (0.09) 0.98 Cognition −0.04(0.06)  0.47 −0.05 (0.06)  0.47 Swallowing −0.10 (0.27)  0.71 0.15(0.19) 0.43 Eye Movement^(b) 0.24 (0.17) 0.18 0.26 (0.12) 0.66 Speech−0.16 (0.15)  0.29 −0.08 (0.15)  0.57 Hearing 0.44 (0.18) 0.02 −0.29(0.15)  0.07 Memory −0.04 (0.13)  0.77 0.05 (0.12) 0.69 Seizures Modelnot stable — — — Notes: ^(a)One additional outlier removed yielding 0.20(0.35) and p-value of 0.57. ^(b)Eye movement score of 5 removed yielding0.05 (0.13) and p-value of 0.67 (CDA105).

Table 3 contains analysis results that include dose as part of themodel. These models provide information about the effect of dose ofdrug. Note the effect of this when looking at hearing. When dose isincluded in the model, the slope for the change over time is no longersignificant.

TABLE 3 Average change over time (in years) for the NPC score and itscomponents adjusted for dose fit to subjects in the Phase 1 studies Avgchange Avg change over time in over time in yrs yrs with I-IND-1 removedNPC score (std. error) p-value (std error) p-value Total Score^(a) 1.79(1.59) 0.27 0.81 (1.12) 0.48 Total Score with 1.50 (1.67) 0.38 0.61(1.18) 0.61 hearing removed^(b) Total Score with 0.93 (1.73) 0.60 0.07(1.21) 0.95 hearing and ABR removed^(c) Ambulation 0.41 (0.43) 0.35 0.19(0.26) 0.47 Fine Motor −0.10 (0.22)  0.65 −0.10 (0.24)  0.69Cognition^(d) 0.13 (0.15) 0.41 0.14 (0.16) 0.38 Swallowing 0.49 (0.70)0.49 0.10 (0.50) 0.85 Eye Movement^(e) 0.16 (0.45) 0.73 0.20 (0.47) 0.68Speech −0.09 (0.39)  0.83 −0.16 (0.38)  0.68 Hearing 0.40 (0.49) 0.430.27 (0.40) 0.52 Memory −0.22 (0.35)  0.59 −0.33 (0.31)  0.30 SeizuresModel not stable — — — Notes: ^(a)One additional outlier removedyielding 1.09 (0.85) with p-value of 0.22. ^(b)One additional outlierremoved yielding 0.91 (0.90) with p-value of 0.32. ^(c)One additionaloutlier removed yielding 0.38 (0.92) with p-value of 0.42.^(d)Additional outliers when I-IND-1 removed; however, model does notconverge when these outliers are removed. ^(e)CDA105 subject 1 yearvalue of eye movement score of 5 also removed yielding 0.30 (0.32) withp-value of 0.37.

7.1.5.4. Comparison of Phase 1 and I-IND Subjects with CorrespondingSubjects in the Natural History Study

To better understand the effect of cyclodextrin, we compared thesubjects in the Phase 1 and I-IND studies with subjects of comparableage in the NIH Natural History study. For this comparison, we limitedthe data set to subjects who had more than a single time point of dataand were between 6 and 26 years of age. We fit a mixed model with time,treatment group and a time by treatment group interaction term in themodel. Table 4 below presents the results from this analysis.

The results presented in Table 4 are consistent with the slower rate ofchange in the Cyclodextrin group compared to the Natural Historypopulation, with the exception of the eye movement, and hearingsubdomains.

TABLE 4 Average change over time separated out by Cyclodextrin use forboth NIH Phase 1, NIH Natural History, and I-IND subjects Avg changeover Avg change over p-value for time in yrs (std. time in yrs (std.difference in error) for Cyclo- error) for Natural slopes between NPCscore dextrin group History study the two groups Total Score 0.74 (0.67)2.53 (0.27) 0.02 Total Score with 0.31 (0.68) 2.36 (0.27) 0.01 hearingremoved Total Score with −0.02 (0.69) 2.23 (0.28) 0.004 hearing and ABRremoved Ambulation 0.001 (0.18) 0.27 (0.07) 0.18 Fine Motor 0.03 (0.10)0.11 (0.04) 0.34 Cognition −0.04 (0.16) 0.31 (0.06) 0.04 Speech −0.16(0.13) 0.09 (0.05) 0.09 Eye Movement 0.22 (0.16) 0.08 (0.06) 0.42Swallowing −0.11 (0.23) 0.20 (0.09) 0.22 Hearing 0.44 (0.16) 0.17 (0.06)0.13 Memory −0.04 (0.12) 0.18 (0.05) 0.10 Seizures NA NA NA

7.1.5.5. Comparison of Phase 1 Subjects with Corresponding Subjects inthe Natural History Study

To better understand the effect of cyclodextrin within the NIHpopulation, we compared the subjects in the Phase 1 study with subjectsof comparable age in the Natural History Study. For this comparison, welimited the data set to subjects who had more than one time point ofdata and were between 6 and 26 years of age. We fit a mixed model withtime, treatment group, and a time by treatment group interaction term inthe model. The table below presents the results from these analyses,which are similar to those in Table 4. Note that the seizures outcome issomewhat unstable with several outliers. Only the full data set resultsare presented. With the exception of eye movement, all rates in theCyclodextrin group are smaller than those observed in the NaturalHistory study.

TABLE 5 Average change over time separated out by Cyclodextrin use forNIH Phase 1 and NIH Natural History subjects Avg change over Avg changeover p-value for time in yrs (std. time in yrs (std. difference inerror) for Cyclo- error) for Natural slopes between NPC score dextringroup History study the two groups Total Score 1.16 (0.68) 2.53 (0.24)0.06 Total Score with 1.06 (0.66) 2.36 (0.24) 0.08 hearing removed TotalScore with 0.86 (0.68) 2.23 (0.24) 0.06 hearing and ABR removedAmbulation 0.21 (0.18) 0.27 (0.06) 0.78 Fine Motor 0.06 (0.11) 0.11(0.04) 0.66 Cognition −0.05 (0.18) 0.31 (0.07) 0.07 Speech −0.10 (0.14)0.09 (0.05) 0.24 Eye Movement 0.32 (0.18) 0.08 (0.06) 0.21 Swallowing0.17 (0.21) 0.20 (0.08) 0.88 Hearing^(a) 0.11 (0.12) 0.17 (0.04) 0.66Memory 0.06 (0.13) 0.18 (0.05) 0.38 Seizures NA NA NA Notes:^(a)Outliers were detected in this model and removed to create smallerslope values in the Natural history group.

7.1.5.6. Analyses that Compare Only Subjects Who have Ever UsedMiglustat

The results presented below include subjects who used miglustat duringthe time period. This includes eight subjects from the Natural historystudy and 14 subjects from the Phase 1 study. We then reran the analysespresented in Table 4 for this population. These results are presented inTable 6 below.

TABLE 6 Average change over time separated out by Cyclodextrin use forNIH Phase 1 and NIH Natural History subjects who reported Miglustat useAvg change over Avg change over p-value for time in yrs (std. time inyrs (std. difference in error) for Cyclo- error) for Natural slopesbetween NPC score dextrin group History study the two groups Total Score0.71 (0.67) 2.58 (0.24) 0.01 Total Score with 0.59 (0.66) 2.39 (0.23)0.01 hearing removed Total Score with 0.37 (0.67) 2.25 (0.24) 0.01hearing and ABR removed Ambulation 0.12 (0.16) 0.26 (0.06) 0.38 FineMotor 0.06 (0.10) 0.08 (0.04) 0.89 Cognition −0.07 (0.21) 0.33 (0.07)0.09 Speech −0.23 (0.14) 0.08 (0.05) 0.05 Eye Movement 0.12 (0.11) 0.05(0.04) 0.55 Swallowing 0.07 (0.22) 0.20 (0.08) 0.58 Hearing 0.13 (0.14)0.19 (0.05) 0.68 Memory 0.06 (0.15) 0.21 (0.05) 0.38 Seizures NA NA NA

Table 7 below presents the results that include only NIH subjects. Thethree subjects from I-IND are removed for this comparison. The resultspresented here presented here are similar to those presented in Table 5above. The difference between the two populations is that the five NIHsubjects in the Natural History study and one NIH subject in the Phase 1study have been removed from the population as these subjects reportedno miglustat use at any point in time.

TABLE 7 Average change over time separated out by Cyclodextrin use forboth NIH Phase 1 and I-IND subjects who reported Miglustat use Avgchange over Avg change over p-value for time in yrs (std. time in yrs(std. difference in error) for Cyclo- error) for Natural slopes betweenNPC score dextrin group History study the two groups Total Score 0.34(0.67) 2.58 (0.27) 0.004 Total Score with −0.13 (0.68) 2.39 (0.27) 0.001hearing removed Total Score with −0.49 (0.69) 2.25 (0.28) 0.001 hearingand ABR removed Ambulation −0.10 (0.17) 0.26 (0.07) 0.06 Fine Motor0.002 (0.10) 0.08 (0.04) 0.48 Cognition −0.04 (0.17) 0.33 (0.07) 0.06Speech −0.27 (0.13) 0.08 (0.05) 0.01 Eye Movement 0.05 (0.11) 0.05(0.04) 0.97 Swallowing −0.21 (0.24) 0.20 (0.10) 0.13 Hearing 0.48 (0.18)0.19 (0.07) 0.15 Memory −0.04 (0.14) 0.21 (0.06) 0.10 Seizures NA NA NA

7.1.6. Further analysis of clinical data

Further analyses were performed on the same NIH clinical trial dataset,but with 4 data points included in the control data set that were notincluded in the analyses described above. These analyses are summarizedin FIGS. 4, 5, 6 and 8.

7.2. Example 2: Standard Analysis of Drug Product

The hydroxypropyl beta-cyclodextrin product used in the Phase I ClinicalTrial described in Example I was Kleptose® HPB (Roquette, France) with aDSa of about 4.34±10%. Standard analyses of two exemplary lots ofKleptose® HPB, as performed by the manufacturer, are shown on thefollowing 8 pages.

Kleptose® HPB Parental Grade (Exemplary Batch 1) Page 1/4 Definition:

HYDROXYPROPYLBETADEX is a partially substituted Poly (hydroxypropyl)ether of betadex.

CAS no: 128446-35-5

EINECS: 420-920-1

INCI: HYDROXYPROPYL CYCLODEXTRIN

Specifications: A) Characters

APPEARANCE White or almost white, amorphous or crystalline powder.

SOLUBILITY Freely soluble in water and in propylene glycol.

B) Identification

IDENTIFICATION-TEST A EP-USP/NF See test

IDENTIFICATION-TEST B EP-USP/NF See test

C) TESTS

INFRA-RED EP-USP/NF Conforms

APPEARANCE IN SOLUTION EP-USP/NF Clear, colourless

CONDUCTIVITY EP-USP/NF 200 μS/cm max.

RELATED SUBSTANCES

-   -   IMPURITY A: BETA CYCLODEXTRIN EP 1.5% max.    -   IMPURITY A: BETA CYCLODEXTRIN USP/NF 1.5% max.    -   IMPURITY B: PROPANE 1, 2 DIOL EP 2.5% max.    -   IMPURITY B: PROPANE 1, 2 DIOL USP/NF 2.5% max.    -   KLEPTOSE® HPB PARENTAL GRADE (EXEMPLARY BATCH 1) Page 2/4    -   SUM OF IMPURITIES OTHER THAN A EP 1.0% max.    -   ANY OTHER IMPURITY USP/NF 0.25% max.    -   TOTAL OF OTHER IMPURITIES USP/NF 1% max.    -   HEAVY METALS EP-USP/NF 20 ppm max.    -   LOSS ON DRYING EP-USP/NF 10.0% max.    -   MOLAR SUBSTITUTION EP-USP/NF 0.40-1.50    -   PROPYLENE OXIDE (**) USP/NF 0.0001%

MICROBIAL CONTAMINATION:

-   -   TOTAL AEROBIC MICROBIAL COUNT EP-USP/NF 100 CPU/g max.    -   TOTAL YEASTS AND MOULD COUNT EP-USP/NF 100 CPU/g max.    -   * Compliance data—Tests not performed    -   * Monitoring plan

Kleptose® HPB Parental Grade (Exemplary Batch 1) Page 3/4

-   -   ESCHERICHIA EP-USP/NF Absence in 10 g.    -   SALMONELLA EP-USP/NF Absence in 10 g.

BACTERIAL ENDOTOXINS EP-USP/NF 10 IU/g max

Typical Values:

MOLAR SUBSTITUTION NOMINAL VALUE 0.62

MOLAR SUBSTITUTION (MS) 0.58-0.68

INTERNAL REQUIREMENTS ON RELATED SUBSTANCES:

-   -   IMPURITY A: BETA CYCLODEXTRIN Internal method (/com) 1.0% max    -   IMPURITY B: PROPANE 1.2 DIOL Internal method (/com) 0.5% max    -   ANY OTHER INPURITY Internal method (/com) 0.1% max

PARTICLE SIZE: Sieve

-   -   RESIDUE ON 315 mic. 20% max    -   RESIDUE ON 100 mic. 50% max

Comments:

Methods used by Roquette may be EP or USP or internal validated methodswhich have been compared to the pharmacopeia monograph.

This substance is suitable for use in the manufacture of parenteralpreparations.

CONFORMITY:

Current pharmacopeia: E.P. (1804) and U.S.P/N.F.

Kleptose® HPB Parental Grade (Exemplary batch 1) Page 4/4

Storage:

STORAGE:

-   -   Preserve in well-closed containers.    -   Hygroscopic product: handle under controlled room conditions.

RETEST DATE:

-   -   3 years in its original packaging.

* Compliance data—Tests not performed

* Monitoring plan

Kleptose® HPB Parental Grade (Exemplary Batch 2) Page 1/4 Definition:

HYDROXYPROPYLBETADEX is a partially substituted Poly (hydroxypropyl)ether of betadex.

CAS no: 128446-35-5

EINECS: 420-920-1

INCI: HYDROXYPROPYL CYCLODEXTRIN

Specifications: A) CHARACTERS

APPEARANCE White or almost white, amorphous or crystalline powder.

SOLUBILITY Freely soluble in water and in propylene glycol.

B) IDENTIFICATION

IDENTIFICATION-TEST A EP-USP/NF See test

IDENTIFICATION-TEST B EP-USP/NF See test

C) TESTS

EP-USP/NF Conforms

INFRA-RED

APPEARANCE IN SOLUTION EP-USP/NF Clear, colourless

CONDUCTIVITY EP-USP/NF 200 μS/cm max.

RELATED SUBSTANCES

IMPURITY A: BETA CYCLODEXTRIN EP-USP/NF 1.0% max.

IMPURITY B: PROPANE 1, 2 DIOL EP-USP/NF 0.50% max.

ANY OTHER IMPURITY EP-USP/NF 0.1% max.

TOTAL OF OTHER IMPURITIES EP-USP/NF 1.0% max.

HEAVY METALS EP-USP/NF 20 ppm max.

LOSS ON DRYING EP-USP/NF 10.0% max.

KLEPTOSE® HPB Parental Grade (Exemplary Batch 2) Page 2/4

MOLAR SUBSTITUTION EP-USP/NF 0.40-1.50

PROPYLENE OXIDE (**) EP-USP/NF 1 ppm max.

* Compliance data—Tests not performed

-   -   * Monitoring plan

KLEPTOSE® HPB Parental Grade (Exemplary Batch 2) Page 3/4

-   -   MICROBIAL CONTAMINATION:

TOTAL AEROBIC MICROBIAL COUNT EP-USP/NF 100 CPU/g max.

TOTAL YEASTS AND MOULS COUNT EP-USP/NF 100 CPU/g max.

ESCHERICHIA EP-USP/NF Absence in 10 g.

SALMONELLA EP-USP/NF Absence in 10 g.

BACTERIAL ENDOTOXINS EP-USP/NF 10 IU/g max.

TYPICAL VALUES:

MOLAR SUBSTITUTION NOMINAL VALUE 0.62

MOLAR SUBSTITUTION (MS) 0.58-0.68

PARTICLE SIZE: Sieve

-   -   RESIDUE ON 315 mic. 20% max    -   RESIDUE ON 100 mic. 50% max

Comments:

Methods used by Roquette may be EP or USP or internal validated methodwhich have been compared to the pharmacopeia monograph.

This substance is suitable for use in the manufacture of parenteralpreparations.

Conformity:

Current pharmacopeia: E.P. (1804) and U.S.P/N.F.

KLEPTOSE® HPB Parental Grade (Exemplary Batch 2) Storage:

-   -   Preserve in well-closed containers.    -   Hygroscopic product: handle under controlled room conditions.

RETEST DATE:

-   -   3 years in its original packaging.    -   * Compliance data—Tests not performed    -   ** Monitoring plan

7.3. Example 3: HPLC Separation of Hydroxypropyl Beta-CyclodextrinMixtures

Various chromatographic methods were used to assess the complexity ofthe cyclodextrin mixture in a commercially available parenteral gradehydroxypropyl beta-cyclodextrin pharmaceutical composition, Kleptose®HPB, batches of which were used in a phase I clinical trial.

7.3.1. CD-Screen Column

The HPLC method from European Pharmacopeia monograph number 1804(Hydroxypropylbetadex) (revised Jan. 1, 2009) was used to separatecomponents in commercial Kleptose HBP® (Roquette).

HPLC conditions: Stationary phase: CD-Screen, particle size 5 μm(ChiroQuest), Column: 1=250 mm, Ø=4.0 mm; temperature: 30° C. Mobilephase: mobile phase A: water; mobile phase B: water:methanol (10:90V/V). Flow rate: 1.0 mL/min; Detection: Alltech 3300 evaporativelight-scattering detector; carrier gas: nitrogen; flow rate: 1.5 L/min;evaporator temperature: 70° C.; Injection: 20 μL.

Gradient Program (European Pharmacopeial Method):

TABLE 8 Time (min) Mobile phase A (% V/V) Mobile phase B (% V/V) 0-5 5248  5-15 52 → 0 48 → 100 15-20  0 100 

FIG. 9 depicts the results of Kleptose® HBP (DS_(a)=4.2) hydroxypropylbeta-cyclodextrin in the CD-Screen HPLC method. Species eluted in theorder of increasing DS. Unsubstituted beta-cyclodextrin eluted at about5 min, with monosubstituted hydroxypropyl beta-cyclodextrin eluting atabout 6 min. FIG. 10 depicts comparative chromatograms of hydroxypropylbeta-cyclodextrins using CD-Screen column with methanol and acetonitrilesolvent gradients (Gradient with methanol: 0 min 30% B, 40 min 100% B;Gradient with acetonitrile: 0-5 min 18% B, 25 min 40% B. Other methodparameters remained unchanged). The retention times varied depending onthe solvent strength and polarity of the mobile phase.

Mass spectrometry detection method: Agilent 1260 HPLC with 6460 TripleQuadrupole mass spectrometer. Agilent Jet Stream electrospray ionization(ESI) source, negative mode, m/z 500-3000; fragmentor voltage: 35 V,Source parameters: gas temperature: 300° C., gas flow: 13 L/min,nebulizer: 60 psi, sheath gas flow: 11 L/min, sheath gas temperature(heater): 400° C., capillary voltage: 3500 V. Ammonium formate buffer(0.1 M, pH=6.0) was applied instead of water in the mobile phases forthe HPLC-MS measurements.

FIG. 11 shows the extracted ion chromatogram of the sample from theHPLC-MS analysis. BCD=unsubstituted beta-cyclodextrin; DSx=hydroxypropylbeta-cyclodextrins with DS of x. For example, “DS3” refers tohydroxypropyl beta-cyclodextrins having DS=3.

7.3.2. Reversed Phase C18 Chromatography

Kleptose® HBP (Roquette) (same batch as used in Section 7.3.1) wasanalyzed. An analytical column (4 mm×250 mm) was filled with LiChroprepRP18 silica gel. One set of conditions tested included the following:Stationary phase: LiChroprep RP18 silica gel, particle size 25-40 μm(Merck), Column: 1=250 mm, └=4.0 mm; temperature: 30° C. Mobile phase:mobile phase A: water; mobile phase B: water:methanol R (10:90 V/V). Thegradient program used the following conditions: 0 min at 10% mobilephase B gradient to 20 min at 100% mobile phase B. Flow rate: 1.0mL/min; Detection: Alltech 3300 evaporative light-scattering detector;carrier gas: nitrogen; flow rate: 1.5 L/min; evaporator temperature: 70°C.

FIG. 12 shows a typical HPLC chromatogram of Kleptose® HPB (DS_(a)=4.2)on LiChrosphere C18 stationary phase. FIG. 13 shows comparativechromatograms of hydroxypropyl beta-cyclodextrins using LiChrosphere C18column with methanol and acetonitrile solvent gradients. (Gradient withmethanol: 0 min 10% B, 15 min 30% B, 40 min 80% B. Gradient withacetonitrile: 0 min 5% B, 40 min 80% B. The other parameters of themethod were not changed.)

Mass spectrometry conditions as described in Section 7.3.1 were used.FIG. 14 shows the extracted ion chromatogram of hydroxypropylbeta-cyclodextrins having different DS from the HPLC-MS analysis.

7.3.3. Hydrophilic Interaction Liquid Chromatography (HILIC)

Hydrophilic interaction liquid chromatography (HILIC) uses hydrophilicstationary phases with eluents typically used in reverse phasechromatography. The approach uses liquid-liquid partition chromatographyprinciples such that analytes may elute in order of increasing polarity.The method described herein used an amino column which containedaminopropyl groups bound to the surface of the silica gel.

Kleptose® HBP (Roquette) (same batch as used in Section 7.3.1) wasanalyzed. HPLC conditions: Stationary phase: Nucleosil NH2, particlesize 5 μm (Macherey Nagel), Column: 1=250 mm, 0=4.0 mm; temperature: 30°C. Mobile phase: mobile phase A: acetonitrile—water (80:20 V/V); mobilephase B: water. Flow rate: 1.0 mL/min; Detection: Agilent 385evaporative light-scattering detector; carrier gas: nitrogen; flow rate:1.2 L/min; evaporator temperature: 50° C.; nebuliser temperature: 30° C.

FIG. 15 shows the separation of hydroxypropyl beta-cyclodextrins usingthe HILIC method on a Nucleosil NH2 column.

Mass spectrometry conditions were the same as those described in Section7.3.1. FIG. 16 shows the extracted ion chromatogram of hydroxypropylbeta-cyclodextrins having different DS from the HPLC-MS analysis.BCD=unsubstituted beta-cyclodextrin; DSx=hydroxypropylbeta-cyclodextrins with DS of x. For example, “DS3” refers tohydroxypropyl beta-cyclodextrins having DS=3. On this HPLC method, thehigher DS substituted hydroxypropyl beta-cyclodextrins eluted first.

7.3.4. Silica Gel Chromatography

Kleptose HBP® (Roquette) (same batch as used in Section 7.3.1) wasanalyzed. HPLC method: Stationary phase: LiChrosphere Si-60, particlesize 5 μm (Merck), Column: 1=250 mm, 0=4.0 mm; temperature: 30° C.Mobile phase: mobile phase A: acetonitrile—0.1 M ammonium formate pH=7.5(80:20 V/V); mobile phase B: 0.1 M ammonium formate pH=7.5. Flow rate:1.0 mL/min; Detection: Agilent 385 evaporative light-scatteringdetector; carrier gas: nitrogen; flow rate: 1.2 L/min; evaporatortemperature: 50° C.; nebulizer temperature: 30° C.

FIG. 17 shows the separation of the components of hydroxypropylbeta-cyclodextrins using LiChrosphere Si 60 column. In this instance,the sample was spiked with beta-cyclodextrin to facilitate detection.

Mass spectrometry conditions were the same as those described in Section7.3.1. FIG. 18 shows the extracted ion chromatogram of hydroxypropylbeta-cyclodextrins having different DS. BCD=unsubstitutedbeta-cyclodextrin; DSx=hydroxypropyl beta-cyclodextrins with DS of x.For example, “DS3” refers to hydroxypropyl beta-cyclodextrins havingDS=3. On this HPLC method, the higher DS substituted hydroxypropylbeta-cyclodextrins eluted first.

7.4. Example 4: Improved Analytical Methods

7.4.1. Gas Chromatography

The official European Pharmacopeia monograph method for thedetermination of propylene glycol (PG) content in Hydroxypropylbetadexhas a limit of quantification of only approximately 0.5% relative toHPBCD. Accordingly, a more sensitive analytical method was needed, andan improved method with modified sample preparation (compared to theEuropean Pharmacopeia Hydroxypropylbetadex analysis, monograph number:1804, revised Jan. 1, 2009) was developed. This method was used toquantify PG at 0.01% level (relative to HPBCD), a much greatersensitivity than the limit of detection in the European Pharmacopeialmethod.

The conditions of the method were as follows: Apparatus: Gaschromatograph: Shimadzu GC-17A; Detector: Flame ionization detector(FID); Injector: Shimadzu AOC-5000 auto injector; Software: ShimadzuClass-VP 7.4 Version; Gases: Carrier gas: Helium (99.999%), Other gases:Nitrogen (99.999%), Synthetic air (99.999%), Hydrogen (from WhatmanHydrogen generator).

Column: Supelco Supercowax-10 (30 m × 0.32 mm × 1.0 μm) Rate (° C./min)Temperature (° C.): time (min): — 150 0 200 10 40 240 1Propylene glycol retention time ˜6.35 min; internal standard ethyleneglycol retention time ˜7.15 min.

Five calibration points: between 0.1 and 2 mg/mL corresponding to 0.01%,0.02%, 0.05%, 0.1%, 0.2% PG related to HPBCD.

The calibration stock solutions were prepared from solutions ofapproximately 200 mg of propylene glycol, accurately weighed into 10 mLgraduated glass flask and filled up to the mark with purified water(Table 14). To obtain the concentrations listed in the table below anadequate dilution was performed. Once the target concentration (seeTable 14 below) was achieved, 1 mL from this solution, 100 μL internalstandard solution (IST), 500 mg NaCl and 1 mL water were added in a crewcap vial and the solution was extracted with 1 mL dichloromethane (DCM).The samples were harvested from the organic phase and injected directly.

TABLE 14 Preparation of GC calibration samples Stock Sample ExtractionSample solution Dilution preparation with IST Target 250 mg Not Not Notconcentra- ethylene applicable applicable applicable tion: 50 glycol(EG)/ mg/mL 5 mL water KAL1 200 mg PG/ 50 μL/ 100 μL IST + 1 1 mL TargetPG 10 mL water 10 mL water mL KAL 1 + 500 DCM concentra- mg NaCl + 1 mLtion: 0.1 water mg/mL KAL2 200 mg PG/ 100 μL/ 100 μL IST + 1 1 mL TargetPG 10 mL water 10 mL water mL KAL 2 + 500 DCM concentra- mg NaCl + 1 mLtion: 0.2 water mg/mL KAL3 200 mg PG/ 250 μL/ 100 μL IST + 1 1 mL TargetPG 10 mL water 10 mL water mL KAL 3 + 500 DCM concentra- mg NaCl + 1 mLtion: 0.5 water mg/mL KAL4 200 mg PG/ 500 μL/ 100 μL IST + 1 1 mL TargetPG 10 mL water 10 mL water mL KAL 4 + 500 DCM concentra- mg NaCl + 1 mLtion: 1.0 water mg/mL KAL5 200 mg PG/ 1 mL/ 100 μL IST + 1 1 mL TargetPG 10 mL water 10 mL water mL KAL 5 + 500 DCM concentra- mg NaCl + 1 mLtion: 2.0 water mg/mL

The calibration solution KAL2 was used to determine system suitabilityby performing five parallel measurements (Requirements: RSD <5%—RSD ofthe area ratio of PG and EG).

Extraction blank sample: 1 mL DCM was added to 0.1 mL IST solution, 500mg NaCl and 2 mL distilled water then stirred vigorously for 0.5 min,and left to stay. After the phases were separated, about 0.2 mL of theDCM phase was put into the vial.

Sample preparation: 1 mL DCM was added to 1.0 g of the HPBCD sample, 0.1mL IST solution, 500 mg NaCl and 2 mL distilled water in a crew cap vialand stirred vigorously for 0.5 min, and left to stay. After the phaseswere separated, about 0.2 mL of the DCM phase is put into the vial.Representative chromatogram of Kleptose HPB® (Roquette) samplecontaining ˜0.18% PG is depicted in FIG. 25.

Propylene glycol (PG) content of HPBCD samples was calculated byplotting a calibration curve displaying the propylene glycol/ethyleneglycol (PG/EG) weigh-in concentration ratios (in mg/mL) as a function ofPG/EG peak areas. In the HPBCD samples, an unknown parameter was the PGcontent that was derived from the other three factors and equation ofthe calibration curve.

The suitability of the method to separate monopropylene glycol from itsdi- and tri-substituted derivatives is shown in FIG. 26. Theoversubstituted glycols showed several peaks and eluted later due to thehigher boiling points. Di- and tripropylene glycol content was tested inthe starting material and in the final samples.

The linearity of the method was proven by testing five samples of PGconcentration between 0.1 and 2 mg/mL corresponding to 0.01%, 0.02%,0.05%, 0.1%, 0.2% PG related to HPBCD as described in Table 14. Eachsample was analyzed in triplicate to assess the precision of the method.Table 15 summarizes experimental data of the precision and linearityanalysis. Linearity curve of the calibration set is depicted in FIG. 27.

TABLE 15 Experimental and statistical evaluation of method precision andlinearity KAL1 KAL2 KAL3 KAL4 KAL5 PG/EG PG/EG PG/EG PG/EG PG/EG Sample(area) (area) (area) (area) (area) Slope regression I 0.1372 0.32520.7359 1.6351 2.9511 7.6382 0.9966 II 0.1520 0.3168 0.7052 1.5245 3.22397.6551 0.9988 III 0.1688 0.2839 0.6601 1.4519 2.9009 7.2976 0.9986average 0.153 0.309 0.700 1.537 3.029 stdev 0.016 0.022 0.038 0.0920.180 RSD 10.4 7.1 5.4 6.0 5.9 PG = propylene glycol; EG = ethyleneglycol; RSD = relative standard deviation.The data in Table 15 showed that the method was linear in the 0.01-0.2%PG content range.

7.4.2. Analytical HPLC

Residual BCD (unsubstituted beta-cyclodextrin) content, HPBCD fractionwith degree of substitution of 1, and sum of cyclodextrin (CD) relatedimpurities other than BCD were determined with the HPLC method inEuropean Pharmacopeia 7.8 (Hydroxypropylbetadex, monograph number: 1804,revised Jan. 1, 2009) and as described in Section 7.3.1. Thedistribution fingerprints of the substances were recorded with the samemethod as well.

7.4.3. NMR

The average degree of substitution (DS_(a)) was calculated from theratio of the signal from the three protons of the methyl group in thehydroxypropyl group and the signal from the proton attached to the C1carbon (anomeric proton) of the anhydroglucose units from ¹H-NMRspectrum.

The peak areas of the doublet from the methyl groups at ˜1.2 ppm (A) andof the signals of the anomeric protons between +5 ppm and +5.4 ppm (B)were measured from the ¹H NMR. An exemplary spectrum is shown in FIG.28. As a reference, the peak area of the anomeric protons was set to 7.0because 7 protons provide this peak in beta-cyclodextrin derivatives.Following assignment of the reference peak, the average degree ofsubstitution was calculated using the expression: DS_(a)=A/3.

7.4.4. Cholesterol Solubilization Assay

In vitro cholesterol solubilization assays were performed as follows. Ahydroxypropyl beta-cyclodextrin mixture test solution in distilled wateris stirred at room temperature, whereupon an excess amount ofunesterified cholesterol is added, such that an amount of thecholesterol remains undissolved. After 24 hours, the solids are filteredaway, and the cholesterol present in solution is measured by HPLCmethod.

HPLC conditions: Analytical column: Nucleosil 120, C8, 5 μm, 100 mm×4.0mm (Macherey Nagel); Column temperature: 40° C.; Mobile phase:Acetonitrile:water=78:22; Flow rate: 1.5 mL/min; Injection volume: 20μL; Detector: UV 210 nm; Stop time: 5 min.

A stock solution of cholesterol is prepared by weighing and transferring10 mg cholesterol in to 10 mL of acetonitrile/isopropanol (75:25). Areference solution of cholesterol is prepared by diluting the stocksolution by ten-fold with the HPLC mobile phase (to give a concentrationof 0.1 mg/mL).

The samples for the solubility experiments are diluted after filtrationtwo-fold with acetonitrile, and additional HPLC mobile phase is used forfurther dilution, if needed.

The concentration of dissolved cholesterol is determined with thefollowing equations:

Cholesterol concentration (mg/mL)=(Areas/Area_(R))×Conc_(R)

-   -   Area_(S)=peak area of Cholesterol from the chromatogram of        Sample Solution    -   Area_(R)=peak area of Cholesterol from the chromatogram of        Reference Solution    -   Conc_(R)=concentration of SBECD-WS in the Calibration Standard        solution (mg/mL).

7.5. Example 5: Electrospray MS Analysis

As discussed in Example 2 above, Kleptose® HPB has an average molarsubstitution of 0.58-0.68 (DS_(a) 4.06-4.76), with two representativebatches having an average molar substitution of 0.62 (DS_(a) 4.34).Trappsol® Cyclo™, a hydroxypropyl beta-cyclodextrin compositionavailable from a different manufacturer, has a higher reported averagemolar substitution of about 0.91 (DS_(a) 6.37).

Electrospray mass spectrometry analysis was performed on commerciallyavailable samples of Kleptose® HPB and Trappsol® Cyclo™ (CTD Holdings,Inc.) (“Trappsol®”) by two different laboratories.

The methods used by the first laboratory were as follows. About 50 μg ofhydroxypropyl beta-cyclodextrin (“HPBCD”) sample was dissolved in 1 mLof 1% formic acid in 80% methanol in water. This HPBCD solution wasinfused into an API-4000 mass spectrometer (Applied Biosystems).Positive ion electrospray mode was applied for MS scanning from m/z 1100to 2000. The MS spectra (10-15 average scans) were recorded. Each signalheight of propylene oxide addition products of β-cyclodextrin wasmeasured and the propylene oxide adduct population of HPBCD wascalculated from the sum of individual signal heights. Analyst 1.51software (Applied Biosystems) was used for the MS operation. As shown inFIG. 29A-B, electrospray MS data from the first lab demonstrates thatthe differences in average molar substitution are caused by markedlydifferent degrees and distributions of hydroxypropyl substitutions inKleptose® HPB (FIG. 29A) and Trappsol® (FIG. 29B). Numbers have beenadded to the spectra to identify the number of hydroxypropyl moieties ineach peak.

The methods used by the second laboratory were as follows. Samples wereprepared at 1 mg/mL in water and diluted to ˜5 μM in 1:1water:acetonitrile for electrospraying. Ions were formed using anAgilent Nanospray source with a direct infusion rate of 600 nL/min(sheath gas at 150° C. and flow rate of 5 L/min with a Vcap potential of1500 V). Potentials in the interface between the ESI source and ionmobility drift tube were adjusted for optimal signal intensities.Spectra were accumulated for 3 min at a single potential across thedrift tube and clearly show ion mobility separations between multipleclasses of ions in the electrospray plume. The analyzer used was theAgilent 6560, a linear, low field ion mobility mass spectrometer, andcan be considered a substantially modified version of an Agilent Q-TOFaccommodating an IM drift tube at the MS sampling orifice employing ionfunnel technology. Electrospray MS data from the second laboratory,shown in FIG. 30A-B, confirms the difference in substitution fingerprint(compare FIG. 30A (Kleptose® HPB) and 30B (Trappsol®)). The data alsoconfirm that electrospray MS is sufficiently robust an analytical toolthat it can be used routinely by different labs to reproduciblyfingerprint hydroxypropyl beta-cyclodextrin compositions.

FIG. 31A-C compare electrospray MS data from three different lots ofKleptose® HPB, performed by two different labs (FIGS. 31A and 31B,second laboratory; FIG. 31C, first laboratory), and demonstrates thatthe substitution fingerprint is nearly identical between lots. The lowlot-to-lot variability in substitution fingerprint is consistent withthe observation that the average molar substitution was identicalbetween two exemplary lots of Kleptose® HPB, as discussed in Example 2above. FIG. 32A-B present electrospray MS spectra from two differentlots of Trappsol®, by two different laboratories (FIG. 32A, firstlaboratory; FIG. 32B, second laboratory) using the same conditions aswere used to generate the Kleptose data shown in FIGS. 31A and 31B, anddemonstrate that there is significant lot-to-lot variability in theTrappsol® substitution fingerprint.

FIG. 33A-B show electrospray MS spectra from the second laboratory inwhich the Y axis has been expanded as compared to FIGS. 29-32 to showpeaks between 1090 and 1230 m/z. FIG. 33A is the spectrum obtained fromTrappsol®. FIG. 33B is the spectrum obtained from Kleptose® HPB. Thespectra show that there are significant levels of propylene glycol inTrappsol® (propylene glycol peaks are labeled in FIG. 33A), but not inKleptose® HPB. Kleptose® HPB has a small but detectable amount ofunsubstituted cyclodextrin, shown by the peak labeled in FIG. 33B.

FIG. 34A-B show the further differences between Kleptose® HPB andTrappsol® samples: 1) DS are significantly different between the twosamples. Trappsol® shows condensation reactions that must include bothaxial and equatorial hydroxyls; 2) Principal ions of both samples areNH4⁺ adduction from ammonia present in the solids. MH⁺ ions are alsopresent for each DS; 3) Isotope clusters for each of the major ionsshows doubly charged homo-dimers of the ammonium adducts. In the case ofTrappsol® doubly charged dimers of the protonated ions are seen as well;4) Both materials show doubly charged homo-dimers from adduction of botha proton and ammonium; 5) Doubly charged hetero-dimers, noted for thecase of DS6-DS7 at m/z 1530, are formed in both materials but are farmore intense in Trappsol® than in Kleptose® HPB; 6) All of thesedifferences were maintained at 2.5 μM concentrations in 80% ACN. Thisindicates the dimers have strong intermolecular associations.

FIG. 35A-B show additional differences between Kleptose® HPB andTrappsol® samples are in the form of triply charged dimers of both homoand hetero-origin. These show greater intensity in Trappsol® and arevirtually absent in Kleptose® HPB.

In summary, electrospray MS analysis demonstrates significantdifferences in the substitution fingerprint of the hydroxypropylbeta-cyclodextrin composition used in the phase I clinical trialdescribed in Example 1, Kleptose® HPB, as compared to the substitutionfingerprint of a different hydroxypropyl beta-cyclodextrin compositionthat is commercially available, Trappsol® Cyclo™. Kleptose® HPB has lowlot-to-lot variability in the substitution fingerprint, and low levelsof impurities, notably propylene glycol. In contrast, Trappsol® exhibitshigh lot-to-lot variability in its substitution fingerprint andsignificantly higher levels of propylene glycol, a presumed ototoxin.Trappsol® also exhibits triply charged cyclodextrin dimers of both homoand hetero-origin, which are absent in Kleptose® HPB.

7.6. Example 6: Purification of Hydroxypropyl Beta-CyclodextrinCompositions

Three purification methods (complexation, precipitation, and adsorption)were investigated for their ability to further reduce propylene glycol(PG) and unsubstituted beta-cyclodextrin (DS=0) impurities in Kleptose®HBP.

7.6.1. Complexation/Association with Organic Compounds

Experiment 5.6.1A: 1.0 g HPBCD (Kleptose HBP® (Roquette)) was dissolvedin water (10 mL). p-Xylene (Trial #5.6.1.1) or toluene (Trial #5.6.1.2)(1.0 mL) was added to the solutions and the mixtures were stirred for 24hrs at room temperature and 1 hr at 5-7° C. The opalescent solutionswere filtered through 0.45 μm cellulose acetate membrane filter and thefiltrates were evaporated at 40° C. to dryness.

Trial #5.6.1.1: 0.7 g; yield: 70%.

Trial #5.6.1.2: 0.8 g; yield: 80%.

Experiment 5.6.1B: 2.0 g HPBCD (Kleptose HBP® (Roquette)) was dissolvedin water (4 mL). D-Limonene (Trial #5.6.1.3, 0.04 mL, ˜10 equivalents)or L-menthol (Trial #5.6.1.4, 0.04 g, ˜10 equivalents) or benzyl alcohol(Trial #5.6.1.5, 0.03 mL, ˜10 equivalents) or cholesterol (Trial#5.6.1.6, 0.1 g, ˜10 equivalents) was added to the solution and themixture was stirred for 72 hrs at room temperature. The solutions werekept at 5° C. for 4 hrs.

Trial #5.6.1.4 and Trial #5.6.1.5 remained clear solutions, noprecipitate was formed.

Trial #5.6.1.3 and Trial #5.6.1.6 formed precipitate. The solutions fromTrial #5.6.1.3 and Trial #5.6.1.6 were each filtered through 0.45 μmcellulose acetate membrane filter and the filtrates were evaporated at40° C. until dryness.

Trial #5.6.1.3: 2.1 g; yield: 105%.

Trial #5.6.1.6: 1.4 g; yield: 70%.

Results for the samples derived from selective complex formation orassociation with small molecules are summarized in Table 9.

TABLE 9 Analysis of HPBCD samples after selective complexation orassociation Trial Trial Trial #5.6.1.1 Trial #5.6.1.3 #5.6.1.6Kleptose ® (p- #5.6.1.2 (D- (L- Test HPB Xylene) (toluene) Limonene)menthol) Propylene 0.18% ND ND ND ND glycol content Unsubsti- 0.60%0.38% 0.54% 0.21% 0.60% tuted β- cyclodextrin content HPBCD 3.68% 3.14%3.25% 2.61% 3.42% DS = 1 content Other 0.23% 0.23% 0.19% 0.14% 0.24%cyclodextrin related impurities ND = not determined.

Trials #5.6.1.1, #5.6.1.2, and #5.6.1.3 showed a decrease inunsubstituted beta-cyclodextrin content compared to commercial Kleptose®HBP. In addition, D-limonene in Trial #5.6.1.3 showed a decrease in thelevels of monosubstituted hydroxypropyl beta-cyclodextrin (“HPBCDDS=1”), and other cyclodextrin-related impurities (see FIG. 19). FIG. 19shows the impurities unsubstituted beta-cyclodextrin (BCD) at retentiontime ˜5 min, monosubstituted hydroxypropyl beta-cyclodextrins (DS-1) atretention time ˜6 min, and hydroxypropyl beta-cyclodextrins having DS=2(DS-2) at retention time ˜7.4 min.

7.6.2. Precipitation

General method: 1.0 g HPBCD (Kleptose HBP® (Roquette)) was dissolved in5 mL or 2 mL solvent (S), and precipitated in 50 mL or 20 mLprecipitating agent (PA). The filtered solid material was rinsed 3 timeswith 3 mL PA. The exception was with Trial #5.6.2.11: 1.0 g HPBCD wasdissolved in water (2 mL) and extracted with chloroform (3×10 mL). Theorganic phases were combined, dried on Na₂SO₄, decanted, and evaporatedat 40° C. until dryness. The preparations for each Trial are summarizedin Table 10.

TABLE 10 Preparation of precipitation samples Solvent AmountPrecipitating Amount Trial # (S) S (mL) agent (PA) PA (mL) Yield (%)Comments 5.6.2.1 methanol 5 acetone 50 92 Solvents and 5.6.2.2acetonitrile 96 precipitating agents 5.6.2.3 chloroform 78 were oftechnical 5.6.2.4 2 acetone 95 grade; the filtered 5.6.2.5 acetonitrile98 solids were dried in 5.6.2.6 chloroform 98 air, at room 5.6.2.7 wateracetone 99 temperature 5.6.2.8 acetonitrile 99 5.6.2.9 chloroform 3 × 1096 extraction 5.6.2.4.2 methanol acetone 50 90 Solvents and 5.6.2.5.2acetonitrile 94 precipitating agents 5.6.2.7.2 water acetone 84 were ofHPLC or GC 5.6.2.8.2 acetonitrile 91 quality; the filtered 5.6.2.10methanol acetone 20 92 solids were dried 5.6.2.11 water chloroform NDunder vacuum, at room temperature ND = not determined.

Analytical results of solvent precipitation experiments are summarizedin Table 11.

TABLE 11 Comparative analysis of the HPBCD samples purified byprecipitation Kleptose ® Trial Trial Trial Trial Trial Trial Test HPB5.6.2.10 5.6.2.2 5.6.2.3 5.6.2.7.2 5.6.2.8.2 5.6.2.11 Propylene glycol0.18% 0.015% 0.035% 0.14% <0.01% <0.01% 0.18% β-cyclodextrin 0.60% ND NDND ND ND ND HPBCD DS = 1 3.68% ND ND ND ND ND ND Total other 0.23% ND NDND ND ND ND cyclodextrin related impurities ND = not determined.

As shown in Table 11, precipitation reduced the level of propyleneglycol in the hydroxypropyl beta-cyclodextrin samples. Trials #5.6.2.10,5.6.2.2, and 5.6.2.3 using methanol in combination with acetone,acetonitrile, or chloroform, respectively, as precipitating agents wereable to reduce the propylene glycol levels by 92%, 81%, and 22%,respectively. Trials #5.6.2.7.2 and 5.6.2.8.2 using water in combinationwith acetone or acetonitrile, respectively, as precipitating agentsreduced the propylene glycol levels by >95% (see FIG. 20, comparingTrial #5.6.2.7.2 versus Kleptose HPB®). In comparison, Trial #5.6.2.11using water and chloroform did not significantly change the propyleneglycol levels as compared with the commercial Kleptose HPB®.

7.6.3. Adsorption

7.6.3.1. Clarification with Alumina

2.0 g HPBCD (Kleptose HBP® (Roquette)) was dissolved in methanol (8 mL,Trial #5.6.3.1.1) or ethanol (8 mL, Trial #5.6.3.1.2), and stirred forhalf an hour with alumina (2.0 g, aluminum oxide 90 standardized, Merck)at room temperature. The alumina was filtered out, washed with methanolor ethanol (3×2 mL), and water (3×2 mL). The filtrates (SZ) and thefirst rinsing solvents (M1 [methanol] or E1 [ethanol]) were evaporatedat 40° C. until dryness.

-   -   Trial #5.6.3.1.1: Sample 5.6.3.1.1-SZ: 1.6 g; yield: 80%.    -   Trial #5.6.3.1.1: Sample 5.6.3.1.1-M1: 0.2 g; yield: 10%.    -   Trial #5.6.3.1.2: Sample 5.6.3.1.2-SZ: 1.5 g; yield: 75%.    -   Trial #5.6.3.1.2: Sample 5.6.3.1.2-E1: 0.2 g; yield: 10%.

7.6.3.2. Chromatography on Alumina

Trial #5.6.3.2.1: 2.0 g HPBCD (Kleptose HBP® (Roquette)) was dissolvedin methanol (2 mL), and chromatographed through alumina (10 g, aluminumoxide 90 standardized, Merck) with methanol, flow rate: 3 mL/min, 1min/fraction, 30 fractions. The column was washed with water, flow rate:10 mL/min, 20 mins (W). The following fractions were combined andevaporated at 40° C. until dryness:

-   -   Sample 5.6.3.2.1A: 2^(nd) fraction: 0.5 g, yield: 25%.    -   Sample 5.6.3.2.1B: 3-8th fractions: 1.1 g, yield: 55%.    -   Sample 5.6.3.2.1C: 9-14th fractions: 0.2 g, yield: 10%.    -   Sample 5.6.3.2.1D: 15th-W fractions: 0.2 g, yield: 10%.

Trial #5.6.3.2.2: 5.0 g HPBCD (Kleptose HBP® (Roquette)) was dissolvedin methanol (10 mL), and chromatographed through alumina (200 g,aluminum oxide 90 standardized, Merck) with the following solventgradient: 100% methanol, flow rate: 5 mL/min, 3 min/fraction, 1-30thfractions; 80% methanol, 20% water, 5 mL/min, 3 min/fraction, 31-43rdfractions; 50% methanol-water, 5 mL/min, 3 min/fraction, 44-60thfractions; 100% water, 5 mL/min, 3 min/fraction, 61-70th fractions;washing (W): 100% water, 10 mL/min, 30 min.

The following fractions were combined and evaporated at 40° C. untildryness:

-   -   Sample 5.6.3.2.2A: 0-5th fractions: 0.1 g, yield: 2%.    -   Sample 5.6.3.2.2B: 6-10th fractions: 0.6 g, yield: 12%.    -   Sample 5.6.3.2.2C: 11-16th fractions: 0.5 g, yield: 10%.    -   Sample 5.6.3.2.2D: 17-45th fractions: 1.1 g, yield: 22%.    -   Sample 5.6.3.2.2E: 46-51st fractions: 0.5 g, yield: 10%.    -   Sample 5.6.3.2.2F: 52-56th fractions: 0.9 g, yield: 18%.    -   Sample 5.6.3.2.2G: 57-66th fractions: 0.5 g, yield: 10%.    -   Sample 5.6.3.2.2H: 67-W fractions: 1.3 g, yield: 26%.

7.6.4. Anion Exchange Resin

Trial #5.6.3.3: 1.0 g HPBCD was dissolved in water (10 mL), and the pHwas set between 10-12 (measured by universal pH paper, Merck pH1-14)with 0.1 N NaOH. The solution was stirred with 5.0 g of anion exchangeresin (Purolite, product code: 47111) for 14 hrs. The resin was filteredout, the filtrate was neutralized with cation exchange resin (Purolite,product code: 15131) and treated with charcoal. The solid sample wasisolated by evaporation at 40° C. until dryness to give Sample 5.6.3.3,Yield: 0.9 g, 90%.

Characterization of samples from adsorption

TABLE 12 Comparative analysis of the HPBCD samples purified by selectiveadsorption Kleptose ® Sample HPB 5.6.3.1.1SZ 5.6.3.1.2SZ 5.6.3.2.1A5.6.3.2.1B 5.6.3.2.1C 5.6.3.3 Propylene glycol 0.18% ND ND 0.10% 0.18%ND 0.15% β-cyclodextrin 0.60% 0.13% 0.33% 0.14% 0.12% 0.23% 0.36% HPBCDDS = 1 3.68% 2.11% 2.45% 1.74% 2.16% 4.74% 2.69% Total other 0.23% 0.1% 0.15% 0.06% 0.06% 0.12% 0.19% cyclodextrin related impurities ND = notdetermined.

TABLE 13 Comparative analysis of the HPBCD samples purified by selectiveadsorption (chromatography) Kleptose ® 5.6.3.2.2 Sample HPB A B C D E FG H Propylene glycol 0.18% ND 0.26% 0.06% 0.31% 0.16% 0.05% 0.05% 0.03%beta-cyclodextrin 0.60% <0.05% <0.05% <0.05% <0.05% <0.05% <0.05% <0.05%1.53% HPBCD DS = 1 3.68% <0.05% 0.05% <0.05% <0.05% 0.07% 0.14% 0.49%10.79% Total other 0.23%  0.38% <0.05% <0.05% 0.08% 0.23% 0.20% 0.51%1.18% cyclodextrin related impurities ND = not determined.

A graphical representation of the data in Tables 12 and 13 is depictedin FIGS. 21-24. Fractions A through H correspond to Samples 5.6.3.2.2Athrough 5.6.3.2.2H, respectively. While anion exchange resin followed bycharcoal treatment served to reduce some of the impurities (Trial#5.6.3.3, FIG. 21), aluminum oxide adsorption was found to be effectivein reducing CD related impurity levels (Trial #5.6.3.1, FIG. 22), evenmore so when applied in chromatography (FIG. 23). FIGS. 21-23 show theimpurity unsubstituted beta-cyclodextrin (BCD) at retention time ˜5 min,as well as monosubstituted hydroxypropyl beta-cyclodextrins (DS-1) atretention time ˜6 min, and hydroxypropyl beta-cyclodextrins having DS=2(DS-2) at retention time ˜7.4 min. The eight fractions (A to H)collected in Trial #5.6.3.2.2 are depicted in FIG. 24. Fractions B-Dcontained the target impurities, propylene glycol and BCD, in extremelylow concentrations. BCD was reduced below the quantification limit infractions A-G. Concomitantly, DS-1 HPBCD content was below 0.15% infractions A-F (96% or greater reduction); and CD-related otherimpurities decreased significantly in fractions B-D. Based on thesefindings, Al₂O₃ based chromatography was deemed suitable to efficientlyremove CD related impurities from HPBCD.

7.7. Example 7: Purification on Larger Scale

To prepare for a phase II clinical trial, three purification methods(precipitation, adsorption on alumina, and a combination ofprecipitation and alumina absorption) were investigated for theirability to reduce propylene glycol (PG) and unsubstitutedbeta-cyclodextrin (DS=0) impurities in Kleptose® HBP on a larger scale.Purification was performed on 10 g batches of HPBCD (Kleptose® HBP(Roquette)); precipitation was also performed on a 30 g batch. Resultsobtained from the 10 g batches are set forth in the Tables 16, 17 and18. Results obtained from the 30 g batch is set forth in the Table 19.

7.7.1. Solvent Precipitation

During preparation of 10 g HPBCD, the precipitation was done from waterwith acetone. HPBCD was dissolved in half equivalent water (20 g HPBCDin 10 ml water), poured into five times volume (100 ml) of acetone andwashed thrice with double volume (40 ml) of acetone after decanting. Thedissolution takes 30-60 minutes using ultrasound and stirring. The glueynature of the precipitate was reduced by thrice washing with acetoneprior to filtering. The yield was 87-93%.

At the 30 g scale methanol was used instead water for the dissolution ofHPBCD. 43 g HPBCD was dissolved in 43 ml methanol, and then poured into430 ml acetone. The precipitate was easy to filter after thrice washingwith acetone (43 ml). The yield was 99%. Based on the GC results, usingmethanol during purification was faster and resulted in a better yieldthan using water.

7.7.2. Chromatography on Alumina

The HPBCD was dissolved in methanol to obtain approximately 1 mg/mlsolutions. For example, 10.0 g HPBCD was dissolved in methanol (10 mL),and chromatographed through alumina (180 cm³ column of 220 g aluminapacking) with methanol, flow rate of 5 mL/min, fractions were harvestedevery 10 minutes with 8-20 fractions in total. The eluent was 100%methanol. To manufacture 10 g of purified HPBCD 20-22 g of startingmaterials were used with a 70-75% yield for the pure product.

7.7.3. Combination of Chromatography on Alumina and SolventPrecipitation

For the combination protocol the solvent precipitation as describedabove was followed by above described chromatography on alumina for 10.0g HPBCD batches.

TABLE 16 Analysis of the HPBCD samples purified by solvent precipitation(Batch ID: CYL-4061) Test Analysis Result Identification HPLC, NMRconforms Average degree of substitution NMR 4.2 Unsubstitutedβ-cyclodextrin content HPLC 0.6% HPBCD DS-1 HPLC 3.7% Total othercyclodextrin related HPLC 0.08% impurities Propylene glycol content GC0.01% Di-propylene glycol content GC <0.2% Tri-propylene glycol contentGC <0.2% Cholesterol solubilizing potency HPLC 23 mg/ml (at 50 mg/ml CDconcentration) Solubility (in 100 cm³ solvent, at Methanol: >60 g 25°Celsius) Ethanol: >60 g

TABLE 17 Analysis of the HPBCD samples purified by absorptionchromatography on alumina (Batch ID: CYL-4062) Test Analysis ResultIdentification HPLC, NMR conforms Average degree of substitution NMR NDUnsubstituted β-cyclodextrin content HPLC <0.05% HPBCD DS-1 HPLC 0.1%Total other cyclodextrin related HPLC 0.05% impurities Propylene glycolcontent GC 0.05% Di-propylene glycol content GC <0.2% Tri-propyleneglycol content GC <0.2% Cholesterol solubilizing potency HPLC 27 mg/ml(at 50 mg/ml CD concentration) Solubility (in 100 cm³ solvent, atMethanol: >60 g 25° Celsius) Ethanol: >60 g

TABLE 18 Analysis of the HPBCD samples purified by combination ofabsorption chromatography on alumina and solvent precipitation (BatchID: CYL-4063) Test Analysis Result Identification HPLC, NMR conformsAverage degree of substitution NMR 4.6 Unsubstituted β-cyclodextrincontent HPLC <0.05% HPBCD DS-1 HPLC 0.02% Total other cyclodextrinrelated HPLC <0.05% impurities Propylene glycol content GC <0.01%Di-propylene glycol content GC <0.2% Tri-propylene glycol content GC<0.2% Cholesterol solubilizing potency HPLC 22 mg/ml (at 50 mg/ml CDconcentration) Solubility (in 100 cm³ solvent, at Methanol: >60 g 25°Celsius) Ethanol: >60 g

TABLE 19 Analysis of the HPBCD (30 g) samples purified by combination ofabsorption chromatography on alumina and solvent precipitation (BatchID: CYL-4077) Test Analysis Result Identification HPLC, NMR conformsAverage degree of substitution NMR 4.7 Unsubstituted β-cyclodextrincontent HPLC <0.02% HPBCD DS-1 HPLC 0.12% Total other cyclodextrinrelated HPLC <0.02% impurities Propylene glycol content GC <0.02%Cholesterol solubilizing potency HPLC 22 mg/ml (at 50 mg/ml CDconcentration) Solubility (in 100 cm³ solvent, at Methanol: >60 g 25°Celsius) Ethanol: >60 g

Table 20 compares results from the 3 purification methods.

TABLE 20 Comparison of purification methods combination of absorptioncombination of absorption absorption chromatography on aluminachromatography on alumina solvent chromatography and solventprecipitation and solvent precipitation precipitation on alumina(water-acetone) (10 g) (methanol-acetone) (30 g) Kleptose ® Batch ID:Batch ID: Batch ID: Batch ID: Test HPB (CYL-4061) (CYL-4062) (CYL-4063)(CYL-4077) Identification conforms conforms conforms conforms Averagedegree of 4.34 4.2 ND 4.6 4.7 substitution Unsubstituted β- 0.60% 0.6%<0.05% <0.05% <0.02% cyclodextrin content HPBCD DS-1 3.68% 3.7% 0.1%0.02% 0.12% Total other 0.23% 0.08% 0.05% <0.05% <0.02% cyclodextrinrelated impurities Propylene glycol 0.18% 0.01% 0.05% <0.01% <0.02%content Di-propylene glycol <0.2% <0.2% <0.2% content Tri-propyleneglycol <0.2% <0.2% <0.2% content Cholesterol 23 mg/ml 27 mg/ml 22 mg/ml22 mg/ml solubilizing potency (at 50 mg/ml CD concentration) Solubility(in 100 cm³ Methanol: >60 g Methanol: >60 g Methanol: >60 gMethanol: >60 g solvent, at 25° Ethanol: >60 g Ethanol: >60 gEthanol: >60 g Ethanol: >60 g Celsius)

7.7.4. Effect of Purification on Substitution Fingerprint

Electrospray MS analysis was performed essentially as described inExample 5 (second laboratory) on an aliquot of Batch CYL-4063, the batchpurified by combination of absorption chromatography on alumina andsolvent precipitation (water-acetone), and on an aliquot of the parentlot of Kleptose® HPB. The spectra are compared in FIG. 36A-B, with FIG.36A showing the spectrum from the starting material and FIG. 36B showingthe spectrum from the purified batch. As can be seen, the purificationeliminates unsubstituted beta-cyclodextrin molecules (“DS-0”), nearlyall cyclodextrin molecules with a single hydroxypropyl substitution(“DS-1”), and reduces the concentration of cyclodextrin molecules havingtwo hydroxypropyl substitutions (“DS-2”). The spectra also suggestlittle change in the relative proportions and thus distribution of themore highly substituted hydroxypropyl beta-cyclodextrin species, DS3,DS4, DS5, DS6 and DS7.

We quantified the peak distribution of 14 batches of Kleptose® HPB(“EOxxx”) and the purified Batch CYL-4063. The signal of each peak andthe percentage each peak contributes to the total signal are summarizedin Table 21.

TABLE 21 Quantification of peak distribution m/z m/z m/z m/z m/z Batch1152 1210 1268 1326 1384 ID (DS-0) (DS-1) (DS-2) (DS3) (DS4) EO190 827140791 139700 321271 418999 0.6% 2.8% 9.7% 22.2% 29.0% EO194 0 74463261482 581280 683474 0.0% 3.1% 10.9% 24.1% 28.4% EO197 14510 67730211808 438806 488923 0.8% 3.8% 12.0% 24.9% 27.7% EO199 14595 80072258738 547035 604472 0.7% 3.6% 11.8% 24.9% 27.5% EO230 9783 51565 178426349066 380442 0.7% 3.7% 12.7% 24.9% 27.2% EO234 12101 64237 210693427987 486218 0.7% 3.7% 12.2% 24.7% 28.1% EO240 10871 55903 187803365483 390189 0.7% 3.9% 12.9% 25.2% 26.9% EO242 7678 31064 101220 192348192221 1.0% 4.2% 13.7% 26.1% 26.1% EO253 11527 53739 174134 335106358419 0.9% 4.0% 12.9% 24.8% 26.5% EO265 0 40533 128930 263039 2963420.0% 3.8% 12.1% 24.7% 27.9% EO270 18518 101531 340195 681274 764792 0.7%3.7% 12.3% 24.7% 27.7% EO277 15144 69571 229585 488128 540927 0.8% 3.5%11.7% 24.8% 27.5% EO237 27395 143476 478761 988885 1124832 0.7% 3.6%11.9% 24.6% 28.0% EO245 0 295840 965902 1907408 2345018 0.0% 3.5% 11.4%22.5% 27.6% Mean 0.6% 3.6% 12.0% 24.5% 27.6% SD 0.3% 0.3% 1.0% 1.0% 0.7%CYL- 0 0 258436 1401875 2525377 4063 0.0% 0.0% 3.2% 17.1% 30.9% m/z m/zm/z m/z Batch 1442 1500 1558 1618 Total DS-2/ ID (DS5) (DS6) (DS7) (DS8)Signal DS-1 EO190 312241 154341 50521 0 1446134 3.4 21.6% 10.7% 3.5%0.0% 100.0% EO194 493534 235243 78741 0 2408217 3.5 20.5% 9.8% 3.3% 0.0%100.0% EO197 334509 155845 52450 0 1764582 3.1 19.0% 8.8% 3.0% 0.0%100.0% EO199 417563 202219 67725 8196 2200613 3.2 19.0% 9.2% 3.1% 0.4%100.0% EO230 264898 125560 41141 0 1400879 3.5 18.9% 9.0% 2.9% 0.0%100.0% EO234 329743 152141 49506 0 1732625 3.3 19.0% 8.8% 2.9% 0.0%100.0% EO240 270539 127936 42957 0 1451681 3.4 18.6% 8.8% 3.0% 0.0%100.0% EO242 128433 60981 23856 0 737801 3.3 17.4% 8.3% 3.2% 0.0% 100.0%EO253 254261 119699 44125 0 1351010 3.2 18.8% 8.9% 3.3% 0.0% 100.0%EO265 203297 98477 32960 0 1063578 3.2 19.1% 9.3% 3.1% 0.0% 100.0% EO270530914 241841 82688 0 2761754 3.4 19.2% 8.8% 3.0% 0.0% 100.0% EO277377689 178205 59435 8755 1967438 3.3 19.2% 9.1% 3.0% 0.4% 100.0% EO237769561 363246 123518 0 4019673 3.3 19.1% 9.0% 3.1% 0.0% 100.0% EO2451807732 880668 285778 0 8488345 3.3 21.3% 10.4% 3.4% 0.0% 100.0% Mean19.3% 9.2% 3.1% 0.1% 100.0% 3.3 SD 1.1% 0.7% 0.2% 0.1% 0.1 CYL- 23280431223567 409420 35622 8182339 N/D 4063 28.5% 15.0% 5.0% 0.4% 100.0%

7.8. Example 8: Effects of Purification on Gene Expression Profiles

As discussed in Example 7, purification efforts at large scale weresuccessful in reducing propylene glycol, which is a presumed ototoxin;beta-cyclodextrin molecules having no hydroxypropyl substitutions(DS-0), which are known to form precipitates; and bacterial endotoxin,which is highly inflammatory. However, we observed that absorptionchromatography with alumina, whether used alone or in combination withsolvent precipitation, also effected significant alteration in thecompositional profile, or fingerprint, substantially reducing the amountof DS-1 hydroxypropyl beta-cyclodextrin, and—in the batch analyzed byelectrospray MS, which had been purified by a combination of absorptionchromatography with alumina and solvent precipitation—reducing theamount of DS-2 hydroxypropyl beta-cyclodextrin, with little apparenteffect on the presence and ratios of the more highly substituted speciespresent in Kleptose® HPB(DS3, DS4, DS5, DS6, DS7).

In order to assess the potential pharmacological effects of thissubstantial alteration in compositional fingerprint, we performed geneexpression profiling.

7.8.1. Materials & Methods

7.8.1.1. Cells

GM18453 cells (homozygous for NPC1 mutation) and wild type GM05659 cellswere obtained from Coriell Medical Institute. Cell lines were culturedin 10% FBS, DMEM, and 100 units/ml of penicillin and streptomycin.500,000 cells/well were synchronized with 10 ug/ml mitomycin prior tocyclodextrin treatment. Hydroxypropyl cyclodextrins (HPCDs) weredissolved in PBS at the range of concentrations used in the study (0.1mM to 10 mM).

7.8.1.2. RNA Preparation and Whole Transcriptome Analysis

NPC1 (GM18453) and wild type cells (GM05659) were lysed and RNAextracted using Trizol® reagent (Invitrogen). The cells were thensubjected to DNAse I (Qiagen) treatment. The purity and concentration ofsamples was checked with both Qubit spectrophotometer and Nano DropND-1000 and the RNA integrity (RIN) was evaluated using Agilent 2100Bioanalyzer. Extracted mRNA was enriched using RiboMinus™ Eukaryote kit(Invitrogen) according to manufacturer's instructions. The finalquantity of RNA was 10 μg per reaction. cDNA libraries generated usingthe Clontech SMARTer Stranded RNA-Seq Kit were size-selected in therange of 150-250 bp and sequenced in accordance to the protocol providedby Illumina. Samples were sequenced using the HiSeq 2000 platform with75 bp forward and 35 reverse primers.

7.8.1.3. RNA-Seq Data Analysis

Sequencing of cDNA libraries resulted in 20941134 to 42375128 pairedreads per sample. For greater mapping quality, base reads were trimmedto 50 base pairs. All color-spaced reads were aligned to human referencegenome (Ensembl, release 73) using TopHat v2.1.0 that used Bowtieversion 1.0.0. Values for RPKM (reads per kilobase of transcript permillion mapped reads) for assessing gene expression levels werecalculated with Cufflinks v2.0.2 and raw counts were retrieved withIllumina BaseSpace Core Apps using gene annotations of protein codinggenes downloaded from Ensembl (release 73). Differential expression/foldchanges was estimated on raw counts with edgeR. All programs were usedwith their default parameters with TopHat set to not to find noveljunctions.

Fold changes for each gene was visualized using TIBCO Spotfire (version6.5). A combination of treemap and bar graphs were used to displaydifferential gene expression changes. To view full transcriptome changesfor 10 mM HPCD treatments, bar graph views were found to be mostoptimal.

7.8.1.4. RNA Seq Assay Validation Analysis

Raw RNA Seq base reads were subjected to directed computational analysisof a selected set of cholesterol homeostatic genes to establishresponsiveness to Kleptose® HPB treatment. The selected genes are listedin Table 22 below.

TABLE 22 Selected Cholesterol Genes for Gene Response Analysis inGM18453 and GM05659 Cells Gene ID Classification A1DOB cholesteroltransport ABACA1 cholesterol transport ABCG1 cholesterol transport ABCG2cholesterol transport ACAT cholesterol esterification ANKFY1 lysosomalAPOA1 lipid metabolism APOEC1 lipid metabolism CEL lipid transport CETPcholesterol ester transfer CH25H lipid transport DHCR7 lipoproteinassembly FDFT1 cholesterol synthesis GGPS1 cholesterol synthesis IDI1cholesterol synthesis LIPA lipid metabolism MVD cholesterol synthesisSC4MOL cholesterol synthesis SC5DL cholesterol synthesis SOAT2lipoprotein assembly SREBF2 cholesterol synthesis

7.8.1.5. Metabolic Pathway Analysis

All cyclodextrin cell treatments were analyzed to determine keymetabolic pathways affected in the 18453 NPC1 cell line. Using IngenuityPathway Analysis software, Gene ID, fold change and p-value per gene wasused to generate Volcano scatter plots. These plots reveal upregulatedand down regulated genes for the various treatments. Groups of genes arethen probability weighted for their ability to perturb particularmetabolic pathways(−log 10 (pACCC)) versus their differential expressionlevels(−D log 10(pORA). The probabilistic mapping of gene members likelyto perturb pathways does not necessarily exclude other gene members'involvement in perturbing other pathways.

These data were analyzed in the context of pathways obtained from theKyoto Encyclopedia of Genes and Genomes (KEGG) database (Release73.0+/03-16, March 15) (Kanehisa et al., 2000; Kanehisa et al., 2002),the gene ontology from the Gene Ontology Consortium database (2014 Sep.19) (Ashburner et al., 2000; Gene Ontology Consortium, 2001), miRNAsfrom the miRBase (Release 21) and TARGETSCAN (TargetScan Release 6.2(updated March 2015)) databases (Griffiths-Jones et al., 2008; Kozomaraand Griffiths-Jones, 2014; Friedman et al., 2009; Grimson et al., 2007),and diseases from the KEGG database (Release 73.0+/03-16, March 15)(Kanehisa et al., 2000; Kanehisa et al., 2002).

Ingenuity Pathway Analysis(Qiagen) scores the pathways using the impactanalysis proposed by (Draghici et al., 2007; Tarca et al., 2009, Khatriet al., 2007). Impact analysis uses two types of evidence: i) theover-representation of differentially expressed (DE) genes in a givenpathway and ii) the perturbation of that pathway computed by propagatingthe measured expression changes across the pathway topology. Theseaspects are captured by two independent probability values, pORA andpAcc, that are then combined in a unique global p-value. The pathwaytopologies, comprised of genes and their interactions, are obtained fromthe KEGG database (Kanehisa et al., 2000; Kanehisa et al., 2010;Kanehisa et al., 2012; Kanehisa et al., 2014).

The first probability, pORA, represents the probability of obtaining anumber of DE genes on the given pathway greater or equal to the oneobserved just by chance (Draghici et al., 2003; Draghici 2011). Let usconsider there are N genes measured in the experiment, with M of theseson the given pathway. Based on a priori selection of DE genes, K out ofM genes were found to be differentially expressed. The probability ofobserving exactly x differentially expressed genes on the given pathwayis computed based on the hypergeometric distribution: Becausehypergeometric is a discrete distribution, the probability of observingfewer than x genes on the given pathway just by chance can be calculatedby summing the probabilities of having 1 or 2 or . . . or x−1 genes onthe pathway:

$\begin{matrix}{{P\left( {{X = \left. x \middle| N \right.},M,K} \right)} = \frac{\begin{pmatrix}M \\x\end{pmatrix}\begin{pmatrix}{N - M} \\{K - x}\end{pmatrix}}{\begin{pmatrix}N \\K\end{pmatrix}}} & (1)\end{matrix}$

To compute the over-representation p-value of obtaining a number of DEgenes on the given pathway greater or equal to the one observed theIngenuity Pathway tool calculates pORA=p (x)=1−p(x−1):

$\begin{matrix}{{{\text{?}\left( {x - 1} \right)} = {{{P\left( {X = 1} \right)} + {P\left( {X = 2} \right)} + \ldots + {P\left( {X = {x - 1}} \right)}} = {\text{?}\frac{\begin{pmatrix}M \\l\end{pmatrix}\begin{pmatrix}{N - M} \\{K - l}\end{pmatrix}}{\begin{pmatrix}N \\K\end{pmatrix}}}}}{\text{?}\text{indicates text missing or illegible when filed}}} & (2)\end{matrix}$

The second probability, pAcc, is calculated based on the amount ofperturbation measured in each pathway. A perturbation factor is computedfor each gene on the pathway using:

$\begin{matrix}{\mspace{79mu} {{{\text{?}(x)} = {1 - {\text{?}\frac{\begin{pmatrix}M \\l\end{pmatrix}\begin{pmatrix}{N - M} \\{K - l}\end{pmatrix}}{\begin{pmatrix}N \\K\end{pmatrix}}}}}{\text{?}\text{indicates text missing or illegible when filed}}}} & (3)\end{matrix}$

In the equation shown, the term ΔE(g) represents the signed normalizedmeasured expression change of gene g, and α(g) is an a priori weightbased on the type of the gene. The second term is the sum of theperturbation factors of all genes u, directly upstream of the targetgene g, normalized by the number of downstream genes of each such gene N(u). The value of β quantifies the strength of the interaction betweengenes g and u. The sign of β represents the type of interaction: plusfor activation like signals, and minus for inhibition like signals.Subsequently, Ingenuity Pathway Analysis calculates the net perturbationaccumulation at the level of each gene Acc(g), as the difference betweenthe perturbation factor PF(g) and the observed log fold-change:

$\begin{matrix}{\mspace{79mu} {{{PF}(g)} = {{{{a(g)} \cdot \Delta}\; {E(g)}} + {\text{?}\text{?}\frac{{PF}(u)}{\text{?}(u)}}}}} & (4) \\{\mspace{79mu} {{{{Acc}\left( g_{i} \right)} = {{{PF}\left( g_{i} \right)} - {\Delta \; {E\left( g_{i} \right)}}}}{\text{?}\text{indicates text missing or illegible when filed}}}} & (5)\end{matrix}$

All perturbation accumulations are computed at the same time by solvingthe system of linear equations resulting from combining the aboveequation (see above) for all genes on the pathway. Once all geneperturbation accumulations are computed, Ingenuity Pathway computes thetotal accumulation of the pathway as the sum of all absoluteaccumulations of the genes. The significance of obtaining a larger totalaccumulation (pAcc) just by chance is assessed through bootstrap.

The two types of evidence, pORA and pAcc, are combined into one finalp-value using Fisher's method. This p-value is then corrected formultiple comparisons using FDR and Bonferroni. Bonferroni is thesimplest and more conservative of the two (Bonferroni, 1935; Bonferroni,1936). It reduces the false discovery rate by imposing a more stringentthreshold on each comparsion weighed by the total number of comparisons.FDR is more powerful at the extent of discovering more false positives(Benjamini and Hochberg, 1995; Benjamini and Yekutieli, 2001). Itensures that the overall percentage of false positives is below thechosen threshold.

7.8.2. Results

7.8.2.1. Assay Validation

Prior to performing full transcriptome analyses, GM18453 and GM05659cells were treated with a range of Kleptose® HPB concentrations (0.1 mMto 10 mM) and effects on selected cholesterol homeostasis-related geneswere assessed.

The results are tabulated in Table 23 below, and graphed in FIG. 37.Results are expressed as fold changes (log(FC)).

TABLE 23 Kleptose ® HPB treatment of GM05659 and GM18453 cells untreatedGM05659 Gene (wild type) untreated 0.1 mM 1.0 mM 10.0 mM Gene IDClassification cells GM18453 GM18453 GM18453 GM18453 A1DOB cholesterol1.723 0.721 0.6332 2.016 2.558 transport ABACA1 cholesterol 1.115 0.4030.345 1.7634 4.569 transport ABCG1 cholesterol 1.316 0.805 0.717 1.233.109 transport ABCG2 cholesterol 1.432 1.222 1.101 1.893 2.467transport ACAT cholesterol 1.742 1.124 1.112 2.371 2.4322 esterificationANKFY1 lysosomal 1.823 0.957 0.786 2.254 2.279 APOA1 lipid 2.672 1.4591.93447 2.7346 4.9437 metabolism APOEC1 lipid 1.217 1.081 0.9996 1.59882.1115 metabolism CEL lipid transport 2.045 1.038 1.006 1.989 3.229 CETPcholesterol 1.789 1.106 1.4803 2.005 3.7633 ester transfer CHOLESTEROLlipid transport 1.643 0.845 1.2246 2.007 1.788 25H DHCR7 lipoprotein1.821 0.677 0.4668 1.634 1.228 assembly FDFT1 cholesterol 1.145 0.7810.8491 0.9572 1.117 synthesis GGPS1 cholesterol 1.509 1.512 1.432 1.6481.589 synthesis IDI1 cholesterol 2.28 1.402 1.227 2.766 3.444 synthesisLIPA lipid 1.607 0.917 0.893 1.339 1.7232 metabolism MVD cholesterol1.7224 1.593 1.5423 1.6772 2.1077 synthesis SC4MOL cholesterol 1.8371.71 1.6632 1.8905 1.9436 synthesis SC5DL cholesterol 2.734 1.893 2.29113.244 4.131 synthesis SOAT2 lipoprotein 1.436 0.533 0.6021 0.9978 1.639assembly SREBF2 cholesterol 1.6044 0.821 0.5299 1.7541 1.9444 synthesis

The results demonstrate dose-dependent effects of Kleptose® HPB on thehomozygous NPC1 cells, and further demonstrate that of the threeconcentrations tested, 1.0 mM Kleptose® HPB is suitable to providemeaningful data.

7.8.2.2. Analysis of Purified Compositions

7.8.2.2.1. Effect on Expression of Pre-Selected Cholesterol HomeostasisGenes

As discussed in Example 7, we found that purification protocols thatinclude adsorption to alumina significantly change the substitutionfingerprint of the hydroxypropyl beta-cyclodextrin mixture. In order toassess how these changes alter the pharmacological effects of thecomposition, we compared the effects on expression of cholesterolhomeostasis genes of (i) the variously purified compositions and (ii)Kleptose® HPB in GM18453 cells, which are homozygous for the NPC1mutation.

To our surprise, we found no difference.

FIG. 38 shows fold changes in expression in GM18453 cells, which arehomozygous for the NPC1 mutation, of the subset of cholesterolhomeostasis genes in which expression was statistically significantlydifferent (p<0.001) upon treatment, for four different compositions: STD(Kleptose® HPB “standard”); AC (Kleptose® HPB purified by aluminachromatography); SP (Kleptose® HPB purified by solvent precipitation);and AP (Kleptose® HPB purified by alumina chromatography & solventprecipitation). See Example 7 for details on the purification protocolsand respective compositions.

None of the purified compositions, at any of the tested concentrations,differed in effect from the parent composition, Kleptose® HPB (“STD”),despite the removal of low DS species in the alumina-purifiedcompositions. It would appear, therefore, that a relevantpharmacological activity of the composition—its effects on cholesterolhomeostasis—resides in the more highly substituted species present inKleptose® HPB, since removal of the unsubstituted beta-cyclodextrinmolecules (DS-0), monosubstituted hydroxypropyl beta-cyclodextrins(DS-1), and substantial reduction in the di-substituted hydroxypropylbeta-cyclodextrins (DS-2) had no discernible effect on the composition'sability to alter expression of cholesterol homeostasis genes in NPC1cells.

7.8.2.2.2. Whole Genome Analysis

In the analysis described above, we assessed changes in expression ofgenes that had been pre-selected for relevance to the primary defectcaused by the NPC1 mutation, and compared effects on expression of onlythat subset of the pre-selected genes whose expression was statisticallysignificantly different upon treatment with hydroxypropylbeta-cyclodextrin. To confirm the observation that removal of low DSspecies has no appreciable effect on relevant pharmacologic activities,and to further explore possible differences between the compositions, weperformed whole genome analyses to identify the biological pathways mostaffected, on a statistical basis, by treatment.

We performed whole genome pathway analyses of NPC1 cells treated with1.0 mM Kleptose® HPB and 1.0 mM of purified batch CYL-4063. As describedin detail in Example 7, CYL-4063 was prepared from Kleptose® HPB by acombination of absorption chromatography on alumina and solventprecipitation. As shown in Table 21 in Example 7, purificationsignificantly reduced the prevalence of low DS species, with only 3.2%of the cyclodextrin species in CYL-4063 having 0, 1, and 2substitutions, collectively, as compared to an average collectivecontent of DS-0, DS-1, and DS-2 of 16.2% in Kleptose® HPB.

As shown in FIG. 39, of the top four pathways identified asstatistically most significantly affected by treatment with 1.0 mMKleptose® HPB, three are also among the top four pathways moststatistically affected by treatment with 1.0 mM CYL-4063: the erbBsignaling pathway, the MAPK signaling pathway, and the GnRH signalingpathway. These results confirm that there is little perturbation in theoverall activity of the hydroxypropyl beta-cyclodextrin mixture on GM18453 cells, despite removal of the low DS species from CYL-4063.

Moreover, we also observed that the steroid biosynthesis pathway, apathway directly affected by the primary defect caused by the NPC1mutation in these cells, is the second most significantly affectedpathway upon treatment with 1.0 mM CYL-4063, but only sixth moststatistically significant pathway affected by treatment with 1.0 mMKleptose® HPB. These results are consistent with the hypothesis that thecomposition's effects on cholesterol homeostasis resides in the morehighly substituted cyclodextrin species, which are present in greaterconcentration in the purified composition.

7.8.2.3. Activity of Fractions Having Different Degrees of Substitution

To assess directly the differential contribution of cyclodextrin specieshaving different degrees of hydroxypropyl substitution, we fractionateda batch of Kleptose® HPB into three pools, respectively having low,medium, and high degrees of hydroxypropyl substitution, and tested theeffects of these pooled fractions on gene expression in NPC1 cells.

7.8.2.3.1. Methods

(a) Fractionation

Fractions were prepared from Kleptose® HPB batch E0245. The distributionof beta-cyclodextrin species in the E0245 starting material is shown inTable 21 (Example 7), excerpted in relevant part in Table 24 below.

TABLE 24 Quantification of peak distribution m/z m/z m/z m/z m/z m/z m/zm/z m/z Batch 1152 1210 1268 1326 1384 1442 1500 1558 1618 Total ID(DS-0) (DS-1) (DS-2) (DS3) (DS4) (DS5) (DS6) (DS7) (DS8) Signal EO245 0295840 965902 1907408 2345018 1807732 880668 285778 0 8488345 0.0% 3.5%11.4% 22.5% 27.6% 21.3% 10.4% 3.4% 0.0% 100.0%

A 15 g sample of Kleptose® HPB (batch E0245) was separated on aCD-screen column essentially as described in Example 5.3.1 above andfractions were collected. FIG. 40 shows chromatograms of variousfractions obtained from the preparative CD-Screen chromatographicseparation, annotated to show the degree of substitution of thechromatographically separated hydroxypropyl beta-cyclodextrin species.

Fraction 2 was chosen as the fraction having low degrees ofsubstitution, “L” (CYL-4103). The fraction having species with mediumdegrees of substitution, fraction “M” (CYL-4104), is a pool of fractions4-15. The fraction having high degrees of substitution, fraction “H”(CYL-4105), is a pool of fractions 16-24. FIG. 41A-D show electrosprayMS spectra of (A) Kleptose® HPB batch E0245, annotated to identify thesignals by degree of hydroxypropyl substitution; (B) the “L” pooledfraction; (C) the “M” pooled fraction; and (D) the “H” pooled fraction.

(b) Expression Profiling

The NPC cell line GM18453 (500,000 cells per well) was treated inparallel with 1.0 mM of “L”, “M”, and “H” fractions. RNA was isolatedfrom each treatment followed by the generation of cDNA libraries inaccordance with prior methods. Sequencing of cDNA libraries resulted in28891287 to 50245721 paired reads per sample. For greater mappingquality, base reads were trimmed to 50 base pairs. All color-spacedreads were aligned to human reference genome (Ensembl, release 73) usingTopHat v2.1.0 that used Bowtie version 1.0.0. RPKM (reads per kilobaseof transcript per million mapped reads) values for gene expressionlevels were calculated with Cufflinks v2.0.2 and raw counts wereretrieved with Illumina BaseSpace Core Apps using gene annotations ofprotein coding genes downloaded from Ensembl (release 73). Differentialexpression/fold changes were estimated on raw counts with edgeR. Allprograms were used with their default parameters with TopHat set to notto find novel junctions. Fold changes for each gene was visualized usingTIBCO Spotfire (version 7.0). A combination of treemap and bar graphswere used to display differential gene expression changes.

For comparison, similar analyses were performed using Kleptose® HPB andunfractionated purified batch CYL-4077 (see Example 7).

7.8.2.3.2. Results

We performed whole genome transcriptome analyses to identify the 10biological pathways most affected, on a statistical basis, by treatmentwith the “L”, “M”, and “H” Fractions.

FIG. 42 shows the 10 biological pathways most affected by treatment ofthe NPC cells with 1.0 mM of the “L”, “M”, and “H” fractions, ranked indescending order of statistical significance. Consistent with ourearlier observations, the “L” fraction did not cause statisticallysignificant changes in the expression of genes in the steroidbiosynthesis pathway. In striking contrast, the steroid biosynthesispathway is statistically the most significantly affected biologicalpathway upon treatment of NPC1 cells with the “M” fraction. The “M”fraction consists primarily of beta-cyclodextrin species having 3, 4, 5and 6 hydroxypropyl substitutions (DS3, DS4, DS5, and DS6). The “H”fraction, which includes primarily DS5, DS6, and DS7, also causeschanges in expression of the genes of the cholesterol biosynthesispathway, with the cholesterol biosynthesis pathway appearing as thesecond most significant pathway.

These results provide direct evidence that the ability to restorecholesterol homeostasis resides in the cyclodextrin species present inKleptose® HPB that have higher degrees of hydroxypropyl substitution.

7.8.2.4. Comparison of Fractions to Unfractionated Compositions

A corollary to our observation that species of cyclodextrin havingdifferent degrees of hydroxypropyl substitution differentially affectgene expression in NPC cells is that the overall pharmacologicalactivity of the composition as a whole must depend on the compositionalfingerprint, that is, on the relative proportions of the differentiallyhydroxypropylated species present in the composition.

We confirmed this hypothesis by comparing gene expression changes on aspecific gene-by-specific gene (that is, on a GeneID) basis aftertreatment of NPC cells with 1.0 mM of either (i) unfractionatedKleptose® HPB, (ii) unfractionated CYL-4077, which had been purifiedfrom Kleptose® HPB by absorption chromatography on alumina and selectivesolvent precipitation, (iii) the “L” fraction, (iv) “M” fraction, or (v)“H” fraction.

We observed that 64% of genes whose expression is significantly affectedby Kleptose® HPB at 1.0 mM are also significantly affected by treatmentwith the purified batch (Batch CYL-4077) at 1.0 mM.

This shared percentage is markedly higher than the percentage of geneswhose expression is significantly affected both by treatment with 1.0 mMKleptose® HPB and by 1.0 mM of any one of the “L”, “M”, and “H”fractions (37%, 43% and 48%, respectively).

Analogously, the 64% shared identity in genes affected both by Kleptose®HPB and CYL-4077 is markedly higher than the percentage of genes whoseexpression is significantly affected both by treatment with 1.0 mMCYL-4077 and 1.0 mM of any one of the “L”, “M”, and “H” fractions (41%,38% and 44%, respectively).

7.8.2.5. Genes Involved in Autophagy

Hydroxypropyl beta-cyclodextrin has been demonstrated to enhanceautophagic clearance of proteolipids aggregates that accumulate in NPCdisease. (Song et al., 2014, J. Biol. Chem. vol. 289(14), pages10211-10222). We therefore analyzed the effects in GM18453 NPC cells oftreatment with 1.0 mM and 10.0 mM Kleptose® HPB, CYL-4077, and each ofthe “H”, “M”, and “L” fractions on the expression of autophagy-relatedgenes.

Table 25 shows all autophagy genes whose change in expression upontreatment was statistically significant (p<0.05), and demonstrates that1.0 mM concentrations had little effect on autophagy-related genes.

TABLE 25 Analysis of Autophagy Genes of GM18453 Cells (1.0 mM)Kleptose ® HPB CYL-4077 “H” “M” “L” Gene fold Gene fold Gene fold Genefold Gene fold ID changes ID changes ID changes ID changes ID changesATG2B −0.995 ATG2B 1.1123 — — — — — —

Table 26 shows all autophagy genes whose change in expression upontreatment was statistically significant (p<0.05) at 10.0 mMconcentrations.

TABLE 26 Analysis of Autophagy Genes of GM18453 Cells (10.0 mM)Kleptose ® HPB CYL-4077 “H” “M” “L” fold Gene fold Gene fold Gene foldGene fold Gene ID changes ID changes ID changes ID changes ID changesATG10 1.23 ATG10 1.045 ATG5 3.11 ATG5 4.332 ATG10 2.7 ATG2A −0.53 ATG120.79 ATG2 −2.65 ATG10 2.705 ATG16L2 0.57 ATG2A −0.53 ATG10 −5.4 ATG1611.294 ATG3 −0.71 ATG2B 1.1023 ATG18 4.5 ATG12 8.34 ATG4A 1.72 ATG3−0.68 ATG101 2 BECN1 2.7 BECN1 0.45 BECN1 0.96 BECN1 7.12 VPS15 3.356VPS15 5.63 VPS13B 9.28 VPS13B −4.793 VPS36 −4.899 VPS36 −4.112 VPS37B−4.897 VPS37B 4.42

The results using 10.0 mM demonstrate Kleptose® HPB and CYL-4077 havesimilar effect on the expression of the genes involved in autophagy, andfurther demonstrate that these effects are contributed by species havinghigher degrees of substitution, with the “L” fraction having the leasteffect on expression of genes involved in autophagy, compared with “H”fraction and “M” fraction.

7.8.3. Discussion

Using gene expression profile experiments, we have demonstrated that theparenteral grade hydroxypropyl beta-cyclodextrin composition, Kleptose®HPB, is capable of restoring expression levels of cholesterolhomeostasis genes in cells homozygous for the NPC1 mutation, andincreasing expression of autophagy-related genes. These data areconsistent with data from the phase I human clinical trial demonstratingthat intrathecal administration of Kleptose® HPB is effective tostabilize or slow progression of symptoms in patients with NPC disease.Correlation with the clinical data demonstrates that the gene expressionassay can provide an in vitro measure of potency.

Electrospray MS demonstrates that Kleptose® HPB is a complex mixture,containing beta-cyclodextrin molecules having different degrees ofhydroxypropylation in reproducible proportions.

The purification process that we have developed to removeprocess-related and other impurities from Kleptose® HPB adventitiouslyremoves beta-cyclodextrin species with low degrees of substitution,altering the compositional fingerprint. Gene expression profileexperiments demonstrated that despite this change in compositionalfingerprint, there was no significant change in the ability of thecomposition to normalize expression of genes in the cholesterolbiosynthesis pathway in cells homozygous for the NPC mutation.

These data suggested that the relevant pharmacological activity iscontributed primarily by species having greater degrees of substitutionthan those eliminated during purification.

We confirmed this inference by fractionating the complex mixture intopools having beta-cyclodextrin species with low (“L”), medium (“M”), andhigh (“H”) degrees of hydroxypropyl substitution, and assessing effectson gene expression in NPC cells. The results demonstrated that the “L”fraction has no apparent effect on expression of genes in thecholesterol biosynthesis pathway or autophagy, whereas the “M” and “H”fractions significantly affect expression of cholesterol biosynthesisand autophagy genes. These experiments further demonstrated that thepharmacological activity of the composition as a whole is a composite ofthe activities separately contributed by beta-cyclodextrin specieshaving different degrees of hydroxypropyl substitution; the overallpharmacological activity depends on the compositional fingerprint, thatis, on the relative proportions of the differentially hydroxypropylatedspecies present in the composition.

Although the pharmacological activity of the composition as a whole is acomposite of the activities separately contributed by beta-cyclodextrinspecies having different degrees of hydroxypropyl substitution,fortuitously, the species that are removed by our purification process,those with low degrees of hydroxypropyl substitution, contribute little,if at all, to the particular activities of the mixture that arepharmacologically relevant to treatment of NPC disease. This discoverywill allow the novel, more highly purified, and compositionally distinctHPBCD composition we have developed to be administered by intrathecal orintracerebroventricular route to the CSF of patients with NPC diseasefor longer periods, with therapeutic effect and increased safety.

7.9. Example 9: Alternative Preparative Fraction Methodology

As discussed in Example 8, three fractions having different averagedegrees of substitution were prepared for use in gene expressionprofiling experiments using a CD-Screen column for chromatographicseparation.

As an alternative, we also prepared fractions of Kleptose® HPB (batchE0245) using an alumina column, essentially as described in Example5.7.2 above. The sample was applied to the alumina column and elutedisocratically with 100% methanol. FIG. 43 shows chromatograms of variousfractions, annotated to show the numerical fractions pooled to producefractions “A”-“F” and “K”, and further annotated to show the degree ofsubstitution of the chromatographically separated hydroxypropylbeta-cyclodextrin species.

The experimental parameters were further optimized to removeunsubstituted BCD and DS1 HPBCD as much as possible from Kleptose® HPB,with the least loss of DS2 and DS3 HPBCD. The details of thepurification methods are described as below:

Identical Parameters for Each Tested Method (Methods II-XI):

-   -   210 g alumina column;    -   30 ml/min flow rate;    -   100 sec/fraction;    -   60 sec delay time.

Only MeOH as Eluent:

Method II. 21 g Kleptose, 30 fractions, eluent: 100% MeOH isocraticelution, yield: 79.4

Method III. 10.5 g Kleptose, 30 fractions, eluent: 100% MeOH isocraticelution, yield: 72.3%

Method IV. 15 g Kleptose, 30 fractions, eluent: 100% MeOH isocraticelution, yield: 77.7

MeOH+water, as eluents:

Method V. 10.4 g Kleptose, 30 fractions, eluent: 100% MeOH isocraticelution (yield: 73.8%)+30 fractions, 100% MeOH isocratic elution (totalyield of the 60 fractions: 75.9%)+30 fractions, 3-step isocratic elution(90, 80, 70% MeOH, 15 min each) with short (100 sec) transition gradientelutions between each step, total yield of the 90 fractions: 83.1%

Method VI. 10.5 g Kleptose, eluent: MeOH+water, 75 fractions, 20 min100->80% MeOH gradient elution, 106 min 80% MeOH isocratic elution,yield: 82.8%

Method VII. Same as Method VI, but 70% MeOH instead of the 80%, yield:91.2%

Method VIII. 10.5 g Kleptose, eluent: MeOH+water, 75% MeOH isocraticelution, 30 fractions, yield: 86.4%

Method IX. 10.6 g Kleptose, eluent: MeOH+water, 70% MeOH isocraticelution, 30 fractions, yield: 89.2%

EtOH (96% purity)+water, as eluents:

Method X. 10.5 g Kleptose, 30 fractions, eluent: 100% EtOH isocraticelution (yield: 27.6%)+30 fractions, 70% EtOH isocratic elution, totalyield of the 60 fractions: 82.1%

Method XI. 10.5 g Kleptose, 60 fractions, eluent: 80% EtOH isocraticelution, yield: 82.3 FIG. 44A-B show chromatograms of the HPBCD mixtureafter different methods of purification. The ratio of the DS2/DS1 in themixture after purification is summarized in Table 27.

TABLE 27 DS2/DS1 Ratio Sample DS1 area DS2 area DS2/DS1 Method II. 790.210187.9 12.893 Method III. 282.8 7836.0 27.706 Method IV. 607.1 10563.817.400 Method V. 537.5 11873.2 22.090 Method VI. 344.7 11678.9 33.885Method VII. 982.1 14857.8 15.129 Method VIII. 318.9 10708.8 33.577Method IX. 729.9 13698.6 18.768 Starting HPBCD 5013.215 16290.0 3.249(for Method II-IX) Method X. 1371.7 14023.1 10.223 Method XI. 274.19445.5 34.464 Starting HPBCD 5161.88 17371.5 3.365 (for Method X-XI)

7.10. Example 10: Phase I Clinical Trial for NPC at 18 Month

Further analyses were conducted on the data from phase I clinical trialfor Niemann-Pick Disease Type C, including efficacy results at 18months. Results are shown in FIGS. 45-50. As summarized in FIG. 45,annualized slope, change from baseline, and responder analysis were usedto analyze the 18 months phase I clinical trial data. From annualizedrate of change, the 18 months treatment results reveal that the HPBCDmixture is a disease modifying therapy (FIG. 46). The HPBCD mixtureshows consistent improvement or stabilization of disease from thebaseline in NPC patients (FIG. 47). The HPBCD mixture treatment alsoshows a greater percentage of responders showing stable or improvingdisease (FIG. 48). The impact of treatment on hearing is primarily inhigh frequency range, and the impact is correctable with hearing aids(FIG. 49). FIG. 50 summarizes the conclusions to date with respect toimpact of treatment on hearing.

EQUIVALENTS AND INCORPORATION BY REFERENCE

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the disclosure.

What is claimed is:
 1. A method for treating Niemann-Pick disease Type Cin a human, the method comprising administering to the human apharmaceutical composition comprising a mixture of hydroxypropylbeta-cyclodextrin molecules, wherein the pharmaceutical composition isadministered intrathecally or intracerebroventricularly in a dosage ofabout 200 mg to about 1500 mg.
 2. The method of claim 1, wherein thedosage is about 500 mg to about 1200 mg.
 3. The method of claim 1,wherein the dosage is about 800 mg to about 1000 mg.
 4. The method ofclaim 1, wherein the mixture of hydroxypropyl beta-cyclodextrinmolecules has an average molar substitution of 0.40 to 0.80.
 5. Themethod of claim 4, wherein the average molar substitution of the mixtureof hydroxypropyl beta-cyclodextrin molecules is 0.48 to 0.72.
 6. Themethod of claim 4, wherein the average molar substitution of the mixtureof hydroxypropyl beta-cyclodextrin molecules is 0.58 to 0.68.
 7. Themethod of claim 1, wherein the pharmaceutical composition isadministered every week, every two weeks, every three weeks, everymonth, every two months, or every three months.
 8. The method of claim1, wherein the pharmaceutical composition is administered every twoweeks.
 9. The method of claim 1, wherein the pharmaceutical compositionis administered intrathecally via a lumbar injection.
 10. The method ofclaim 1, wherein the pharmaceutical composition further comprises apharmaceutically acceptable carrier.
 11. The method of claim 10, whereinthe pharmaceutically acceptable carrier is an aqueous solution.
 12. Themethod of claim 11, wherein the aqueous solution is an isotonic salinesolution.
 13. The method of claim 1, wherein the human is a pediatricpatient or an adult patient.
 14. The method of claim 13, whereinpediatric patient is less than 2 years of age, from 2 years to 6 yearsof age, from 6 years to 15 years of age, or greater than 15 years ofage.
 15. The method of claim 1, wherein the Niemann-Pick disease Type Cis Niemann-Pick disease Type C1 (NPC1) or Niemann-Pick disease Type C2(NPC2).